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Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly

小麦族系中染色体构象捕获测序：一个有价值的基因组组装工具

Axel Himmelbach Axel Himmelbach
IW Ines Walde
MM Martin Mascher
NS Nils Stein

发布: 2018年08月05日第8卷第15期 DOI: 10.21769/BioProtoc.2955 浏览次数: 10899

评审: Rainer MelzerPooja VermaVinay Panwar

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Abstract

Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was measured by amplification of selected ligation junctions corresponding to a small number of genomic loci (‘one versus one’) through semi-quantitative PCR (Dekker et al., 2002). The chromosome conformation capture-on-chip (4C) and chromosome conformation capture carbon copy (5C) technologies then extended 3C to count ligation events in a ‘one versus many’ or ‘many versus many’ manner, respectively. Hi-C (Lieberman-Aiden et al., 2009) finally combined 3C with next-generation sequencing (Metzker, 2010). Here, before religation sticky ends are filled in with biotin-labeled nucleotide analogs to enrich for fragments with a ligation junction in a later step. The Hi-C libraries are then subjected to high-throughput sequencing and the resultant reads mapped to a reference genome, allowing the determination of contact probabilities in a ‘many versus many’ way with a resolution that is limited only by the distribution of restriction sites and the read depth. The first application of Hi-C was the elucidation of global chromatin folding principles in the human genome (Lieberman-Aiden et al., 2009). Similar efforts have since been carried out in other eukaryotic model species such as yeast (Duan et al., 2010), Drosophila (Sexton et al., 2012) and Arabidopsis (Grob et al., 2014; Wang et al., 2015; Liu et al., 2016). Other uses of Hi-C include the study of chromatin looping at high-resolution (Rao et al., 2014; Liu et al., 2016), of chromatin reorganization along the cell cycle (Naumova et al., 2013) and of differences in chromatin organization in mutant individuals (Feng et al., 2014). The tethered conformation capture protocol (TCC) (Kalhor et al., 2011) described here is a variant of the original Hi-C method (Lieberman-Aiden et al., 2009) and was adapted to Triticeae.

Keywords: Tethered conformation capture (构象捕获) search Hi-C (Hi-C) search Physical mapping (物理图谱) search Chromatin interaction (染色质互作) search Triticeae (小麦族) search Sequencing (测序) search Genome assembly (基因组组装) search Scaffolding (支架) search

Background

Any successful Hi-C experiment should reveal the distance-dependent decay of contact probabilities: the frequency by which two loci juxtapose in three-dimensional space is predominantly determined by their distance in the linear genome (Lieberman-Aiden et al., 2009). This observation motivated the application of Hi-C for physical mapping: the number of Hi-C links between pairs of contigs of a whole-genome shotgun assembly can serve as a proxy of the linear distance between them. This distance information can then be fed into graph algorithms to reconstruct the linear order of scaffolds along the chromosomes (Burton et al., 2013; Kaplan and Dekker, 2013). The three-dimensional proximity information obtained from our TCC approach was employed to order and orient BAC-based super scaffolds in the high-quality barley reference genome (Beier et al., 2017; Mascher et al., 2017). For the Emmer genome assembly, the TCC information was used in a similar manner to validate the scaffolds leading to chromosome-scale assemblies (pseudomolecules) (Avni et al., 2017).

TCC is a modified Hi-C approach in which key reactions (marking of the DNA ends and circularization) are performed on a solid phase rather than in solution (Kalhor et al., 2011). In fact, in human cells the tethering approach yielded improved signal-to-noise ratios, thus leading to a better mapping of low-frequency interactions (Kalhor et al., 2011). This observation prompted us to develop a similar protocol for Triticeae, which is based on the isolation of crosslinked nuclei from young leaves (Hövel et al., 2012) followed by TCC (Kalhor et al., 2011) (Figure 1). Briefly, the chromosome conformation is captured in native leaves by chemical crosslinking using formaldehyde (Hövel et al., 2012), which covalently connects proteins to DNA, as well as proteins to each other. Nuclei are purified, and cysteine residues of proteins contained in the chromatin are biotinylated (Kalhor et al., 2011). The DNA is digested with a restriction enzyme (HindIII) followed by immobilization at low density on streptavidin-coated beads via the crosslinked biotinylated proteins (Kalhor et al., 2011). DNA ends are filled-in, marked with biotin and circularized while tethered to the beads (Kalhor et al., 2011). The crosslinks are reversed, ligation junctions are affinity-purified and provided with adapters for Illumina sequencing to discover genuine pairs of intrachromosomal interaction sites, which were initially captured (Kalhor et al., 2011).

Chromosome conformation capture sequence data is analyzed in the context of genome sequence assemblies. If a chromosome-scale reference genome is available, Hi-C (or TCC) reads can be aligned to it and assigned to restriction fragment predicted in silico from the genome sequence. Subsequently, the number of Hi-C reads linking pairs of restriction pairs can be quantified and the counts be aggregated in larger genome windows (e.g., 1 Mb bins) to obtain estimates of the contact probabilities between pairs of genomic loci (Lieberman-Aiden et al., 2009). These contact probabilities can be used to investigate chromatin organization and its interaction with various biological parameters such as stage of the cell cycle (Naumova et al., 2013) or the epigenomic landscape (Zhou et al., 2013). Chromosome conformation capture sequencing can also be used to reconstruct contiguous, chromosome-scale genomic reference sequences from fragmented sequence assembly. The Hi-C/TCC reads are mapped to a sequence assembly composed of unordered scaffolds, and links between scaffolds are counted and used as a measure of genomic distance. The closer two sequence scaffolds are to each other in the linear genome, the higher the number of Hi-C read pairs linking them. This distance information can then be used to derive a linear order of sequence scaffolds and orient adjacent scaffolds relative to each other (Burton et al., 2013; Kaplan and Dekker, 2013). These approaches have been applied in the sequence assembly of insect (Dudchenko et al., 2017) and Triticeae genomes (Avni et al., 2017; Mascher et al., 2017).


Figure 1. Schematic overview of TCC for Triticeae. Leaves of seven days old plants (I) are harvested for chemical crosslinking of the chromatin (II). The chromatin structure is captured by the formation of covalent bonds between proteins (grey ovals) and between proteins and DNA (line). The orange and light blue segments exemplify two HindIII fragments located on a chromosome. Nuclei are purified (III), and proteins are biotinylated (IV, green pentagon) for subsequent tethering of the complexes to a solid phase. DNA is digested (scissors) using the restriction enzyme HindIII (V) and tethered at low density to streptavidin-coated beads (VI, purple). Ends are marked and filled-in using biotin-14-dCTP (VII, red star). By including dGTPαS in the fill-in reaction, a phosphorothioate bond is introduced. Thereby DNA is guarded against Exonuclease III digestion, which is used at a later step to remove biotinylated nucleotides from non-ligated ends (not shown). Filled-in HindIII sites are ligated ‘on-bead’ (VIII), thereby newly creating NheI restriction sites, which are indicating TCC ligation events. The crosslinking is reversed. DNA is purified (IX) and treated with Exonuclease III (not shown) prior to fragmentation (X). The position of primers (grey triangles) used for controlling the marking and ligation of ends (3C control) is indicated (IX). Ends of the fragmented DNA are repaired and tailed with ‘A’ (not shown). DNA fragments with genuine ligation junctions are affinity-purified based on the incorporated biotin (XI) and provided with Illumina adapters (XII, grey lines) for paired-end sequencing. Ligation products are PCR-amplified (XII; arrowheads: position of primer), size-selected (XIII) and quality controlled (QC). The junctions are revealed by paired-end (PE) sequencing using an Illumina HiSeq2500 device (XIV) followed by bioinformatic analysis (XV). The estimated hands-on time is indicated in days (d). The operating period of the Illumina HiSeq2500 instrument is given for 2x 100 cycles sequencing and depends on the chemistry and type of flowcell (days in brackets). The flowchart is adapted from (Kalhor et al., 2011).

Materials and Reagents

Notes:

  1. We tested all components in independent experiments. However, this list does not imply that alternative products from other manufacturers cannot perform just as well.
  2. The use of the published equipment and chemistry is not indicating any competing interest.
  1. Compost soil (multiple vendors)
  2. Pots (16 cm diameter) (multiple vendors)
  3. Aluminum foil (multiple vendors)
  4. 10 µl Sapphire Low Retention Filtertips (Greiner Bio One International, catalog number: 771265 )
  5. 200 µl Sapphire Low Retention Filtertips (Greiner Bio One International, catalog number: 737265 )
  6. 1,000 µl Sapphire Low Retention Filtertips (Greiner Bio One International, catalog number: 750265 )
  7. 0.2 µm filter, Millex GP (Merck, catalog number: SLGP033RS )
  8. 10 ml and 50 ml syringes (multiple vendors)
  9. 50 ml Falcon tubes (Corning, catalog number: 352070 )
  10. Racks for 50 ml Falcon tubes fitting into the desiccator (multiple vendors)
  11. Disposable 10 ml and 25 ml plastic pipettes (multiple vendors)
  12. Pipette fillers (BRAND, catalog number: 25315 )
  13. Paper towels (multiple vendors)
  14. Appropriate protective gear for working with liquid nitrogen (multiple vendors)
  15. Dewar vessel for transport and storage of liquid nitrogen (multiple vendors)
  16. Miracloth (Merck, catalog number: 475855-1R )
  17. Sefar Nitex 03-55/32 (55 µm mesh opening, 32% open area) (Stefan Kastenmüller GmbH)
  18. Tubes (1.5 ml and 2.0 ml; SARSTEDT, catalog numbers: 72.690.001 and 72.695.500 )
  19. Microscopic slides (76 x 26 x 1 mm) (multiple vendors)
  20. Cover slips (multiple vendors)
  21. Cell counting chamber (Neubauer chamber) and glass cover (Celeromics)
  22. Needle (0.9 x 40 mm, HSW FINE-JECT) (Henke-Sass, Wolf, catalog number: 8300025263 )
  23. Slide-A-LyzerTM Dialysis cassette (0.5-3 ml, cutoff 20 kDa) with float buoys (Thermo Fisher Scientific, catalog number: 66003 )
  24. Parafilm (BRAND, catalog number: 701605 )
  25. Magnetic Particle Concentrator (MPC) for 1.5 ml tubes (DynaMag-2 Magnet) (Thermo Fisher Scientific, catalog number: 12321D )
  26. 15 ml conical tubes (Corning, catalog number: 352196 )
  27. Rubber strap or tape
  28. DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) (Thermo Fisher Scientific, catalog number: 12027 )
  29. PCR-tubes, Sapphire (Greiner Bio One International, catalog number: 652250 )
  30. Snap-cap microTUBEs (with AFA-fiber and pre-split septum) (Covaris, catalog number: 520045 )
  31. Disposable scalpels (multiple vendors)
  32. Polystyrene plugs (self-made) of 0.5 to 1.0 cm thickness fitting snugly into the 50 ml plastic tubes
  33. Seeds (provided by the experimenter)
  34. HEPES sodium salt (4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt) (Carl Roth, catalog number: 7020.2 )
  35. Concentrated HCl (Carl Roth, catalog number: 4625.1 )
  36. Sucrose (Carl Roth, catalog number: 9097.1 )
  37. MgCl2•6H2O (Sigma-Aldrich, catalog number: M2670 )
  38. KCl (Honeywell, Fluka, catalog number: 60130 )
  39. Triton X-100 (Carl Roth, catalog number: 3051.3 )
  40. 100% glycerol (Carl Roth, catalog number: 3783.1 )
  41. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 93482 )
  42. 2-mercaptoethanol (14.3 M) (Carl Roth, catalog number: 4227.1 )
  43. 37% (w/v) formaldehyde solution (containing 10-15% methanol as stabilizer) (Sigma-Aldrich, catalog number: 252549 )
  44. Glycine (Carl Roth, catalog number: 3790.1 )
  45. Liquid nitrogen
  46. Aprotinin (Sigma-Aldrich, catalog number: 10820 )
  47. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  48. Pepstatin (Sigma-Aldrich, catalog number: P5318 )
  49. VECTASHIELD mounting medium with DAPI (VECTOR Laboratories, catalog number: H-1200 )
  50. SDS (Sodium Dodecyl Sulfate) (Serva, catalog number: 20765 )
  51. Tris (hydroxymethyl) aminomethane (Tris base) (Carl Roth, catalog number: 5429.3 )
  52. NaCl (Carl Roth, catalog number: 9265.2 )
  53. EZlink Iodoacetyl-PEG2-Biotin (IPB) (Thermo Fisher Scientific, catalog number: 21334 )
  54. 1,4-Dithiothreitol (DTT) (Carl Roth, catalog number: 6908.1 )
  55. HindIII (100 U/µl) (New England BioLabs, catalog number: R0104T )
  56. NheI (10 U/µl) (Thermo Fisher Scientific, catalog number: ER0971 )
  57. MyOne Streptavidin T1 beads (Thermo Fisher Scientific, catalog number: 65601 )
  58. Na2HPO4 (Carl Roth, catalog number: P030.2 )
  59. KH2PO4 (Carl Roth, catalog number: 3904.1 )
  60. 100 mM dATP (Thermo Fisher Scientific, catalog number: R0142 )
  61. 100 mM dTTP (Thermo Fisher Scientific, catalog number: R0172 )
  62. 2'-Deoxyguanosine-5'-(α-thio)-triphosphate, Sodium salt (dGTPαS), 1:1 Mixture of Rp and Sp isomers (Jena Bioscience, catalog number: NU-424S )
  63. Biotin-14-dCTP (Thermo Fisher Scientific, catalog number: 19518018 )
  64. Klenow DNA polymerase, large fragment (10 U/µl) (Thermo Fisher Scientific, catalog number: EP0052 )
  65. UltraPure BSA (50 mg/ml) (Thermo Fisher Scientific, catalog number: AM2616 )
  66. T4 DNA ligase (5 U/µl) with 10x T4 DNA Ligation buffer and 50% PEG 4000 (Thermo Fisher Scientific, catalog number: EL0011 )
  67. RNase A (DNase-free) (MACHEREY-NAGEL, catalog number: 740505 )
  68. Proteinase K (Thermo Fisher Scientific, catalog number: 25530015 )
  69. Phenol:CHCl3:isoamyl alcohol (25:24:1) (Carl Roth, catalog number: A156.2 )
  70. 8-Hydroxyquinoline (Sigma-Aldrich, catalog number: H6878 )
  71. Isoamyl alcohol (Carl Roth, catalog number: 8930.1 )
  72. CHCl3 (Carl Roth, catalog number: 3313.2 )
  73. Glycogen (Thermo Fisher Scientific, catalog number: R0561 )
  74. 100% ethanol (Carl Roth, catalog number: 9065.4 )
  75. Q5 Hot Start High-Fidelity DNA Polymerase (2 U/µl) and 5x Q5 Hot Start buffer (New England BioLabs, catalog number: M0493S )
  76. Primer for biotin incorporation control (barley):
    Primer 1 (5'- ATCTTCATGCGAGGCAGAGT-3')
    Primer 2 (5'- ACCGTTGAACCATCTTCAGG-3')
    Note: The primers are HPLC purified, 0.2 μmol synthesis scale, dissolved in H2O to 100 µM; contact e.g., Sigma-Aldrich for synthesis.
  77. dNTP Set (100 mM solutions) (Thermo Fisher Scientific, catalog number: R0181 )
  78. dNTP Mix (25 mM each) (Thermo Fisher Scientific, catalog number: R1121 )
  79. MinElute Gel Extraction Kit (QIAGEN, catalog number: 28606 )
  80. Bromophenol Blue (Carl Roth, catalog number: A512.2 )
  81. 10x NEBuffer 1 buffer (New England BioLabs, catalog number: B7001S )
  82. UltraPure Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  83. Exonuclease III (100 U/µl) from E. coli (New England BioLabs, catalog number: M0206S )
  84. AMPure XP beads (Beckman Coulter, catalog number: A63881 )
  85. 10x Tango (Thermo Fisher Scientific, catalog number: BY5 )
  86. 100 mM ATP (Thermo Fisher Scientific, catalog number: R0441 )
  87. T4 DNA polymerase (5 U/µl) (Thermo Fisher Scientific, catalog number: EP0062 )
  88. T4 polynucleotide kinase (10 U/µl) (Thermo Fisher Scientific, catalog number: EK0031 )
  89. Klenow DNA polymerase, large fragment (10U/µl) (Thermo Fisher Scientific, catalog number: EP0052 )
  90. Klenow fragment, 3'→5' exo- (5 U/µl) (New England BioLabs, catalog number: M0212L )
  91. Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific, catalog number: 65001 )
  92. DNA LoBind tubes (1.5 ml) (Eppendorf, catalog number: 0030108051 )
  93. TruSeq DNA Single Indexes Set A (Illumina, catalog number: 20015960 )
  94. Library amplification primer:
    Primer 3 (5'-AATGATACGGCGACCACCGAGAT-3')
    Primer 4 (5'-CAAGCAGAAGACGGCATACGA -3')
    Note: The primers are HPLC purified, 0.2 μmol synthesis scale, dissolved in H2O to 100 µM; contact e.g., Sigma-Aldrich for synthesis.
  95. SYBR-Gold (Thermo Fisher Scientific, catalog number: S11494 )
  96. GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific, catalog number: SM0373 )
  97. Glacial acetic acid (Carl Roth, catalog number: 3738.5 )
  98. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
  99. 100% isopropanol (Carl Roth, catalog number: 6752.4 )
Note: Prepare the following solutions (#100-#107) as described in Green and Sambrook (2012).
  1. 1 M Tris-HCl, pH 8.0 (100 ml)
  2. 1 M Tris-HCl, pH 7.4 (100 ml)
  3. 1 M Tris-HCl, pH 7.5 (100 ml)
  4. 0.5 M EDTA, pH 8.0 (100 ml)
  5. 1 M DTT (20 ml)
  6. Phenol:CHCl3:isoamyl alcohol (25:24:1) with 0.1% 8-Hydroxyquinoline
  7. CHCl3:isoamyl alcohol (24:1)
  8. 1 M MgCl2
  9. 1 M HEPES, pH 8.0 (see Recipes) 
  10. 2 M sucrose (see Recipes)
  11. 1 M KCl (see Recipes)
  12. 10% (v/v) Triton X-100 (see Recipes)
  13. Nuclei Isolation Buffer (NIBF) with formaldehyde (see Recipes)
  14. 2 M glycine (see Recipes)
  15. 1 mg/ml Aprotinin (see Recipes)
  16. 1 mg/ml Leupeptin (see Recipes)
  17. 1 mg/ml Pepstatin (see Recipes)
  18. Nuclei Isolation Buffer with protease inhibitors (NIBP) (see Recipes)
  19. Sucrose cushion (see Recipes)
  20. Nuclei Isolation Buffer with 1.5 M sucrose (NIBS) (see Recipes)
  21. 2% (w/v) SDS (see Recipes)
  22. 4 M NaCl (see Recipes)
  23. Wash buffer 1 (see Recipes)
  24. 25 mM IPB (see Recipes)
  25. 10x NEBuffer 2 (see Recipes)
  26. 1x NEBuffer 2 (see Recipes)
  27. HindIII (10 U/ µl) (see Recipes)
  28. Dialysis buffer (TE buffer) (see Recipes)
  29. Phosphate Buffered Saline with Tween 20, pH 7.4 (PBST) (see Recipes)
  30. 25 mM neutralized IPB (see Recipes)
  31. 10 mM dATP (see Recipes)
  32. 10 mM dTTP (see Recipes)
  33. Wash buffer 2 (see Recipes)
  34. Wash buffer 3 (see Recipes)
  35. 10x ligation buffer TCC (see Recipes)
  36. 10 mg/ml BSA (see Recipes)
  37. Extraction buffer (see Recipes)
  38. 25 mg/ml RNase A (see Recipes)
  39. 20 mg/ml Proteinase K (see Recipes)
  40. 5 mg/ml glycogen (see Recipes)
  41. 80% ethanol (see Recipes)
  42. 70% ethanol (see Recipes)
  43. 3 M sodium acetate, pH 5.2 (see Recipes)
  44. EB (see Recipes)
  45. EBT (see Recipes)
  46. Primer 1 + 2 (10 µM each) (see Recipes)
  47. 10 mM dNTP mix (see Recipes)
  48. 2.5 mM dNTP mix (see Recipes)
  49. TLE (see Recipes)
  50. Tween Wash Buffer (TWB) (see Recipes)
  51. 2x Binding Buffer (2x BB) (see Recipes)
  52. 1x Binding Buffer (1x BB) (see Recipes)
  53. 1x ligation buffer (see Recipes)
  54. Primer 3 + 4 (10 µM each) (see Recipes)
  55. 6x loading dye (see Recipes)
  56. 50x TAE (see Recipes)

Equipment

  1. Scissors (multiple vendors)
  2. Ice bucket (multiple vendors)
  3. Borosilicate glass bottles (100 ml) (Laborbedarfshop, catalog number: GT00205 )
  4. Borosilicate glass bottles (250 ml) (Laborbedarfshop, catalog number: GT00206 )
  5. Borosilicate glass bottles (500 ml) (Laborbedarfshop, catalog number: GT00207 )
  6. Borosilicate glass bottles (1,000 ml) (Laborbedarfshop, catalog number: GT00208 )
  7. Borosilicate Erlenmeyer flasks (500 ml) (Laborbedarfshop, catalog number: GT00153 )
  8. P1, 10 µl Finnpipette (Thermo Fisher Scientific, catalog number: 4641040N )
  9. P10, 100 µl Finnpipette (Thermo Fisher Scientific, catalog number: 4641070N )
  10. P20, 200 µl Finnpipette (Thermo Fisher Scientific, catalog number: 4641080N )
  11. P100, 1000 µl Finnpipette (Thermo Fisher Scientific, catalog number: 4641100N )
  12. Mortar with pestle with a rough surface for grinding (about 10 cm diameter) (multiple vendors)
  13. Metal spoon (multiple vendors)
  14. Funnels fitting into 50 ml tubes (multiple vendors)
  15. Sieve or tea strainer (about 8 cm diameter) (multiple vendors)
  16. Greenhouse equipped with automatic shading and supplementary light (sodium halogen lamps)
  17. pH meter (multiple vendors)
  18. Water bath (65 °C) (multiple vendors)
  19. -20 °C freezer (multiple vendors)
  20. -80 °C freezer (multiple vendors)
  21. Vortex (multiple vendors)
  22. Measuring cylinder 100 ml, 1 L (multiple vendors)
  23. Autoclave (multiple vendors)
  24. Analytical laboratory balance 'Quintix' (0,1 mg to 120 g) (Sartorius, catalog number: Quintix® 124-1S )
  25. Precision laboratory balance 'Cubis' (10 mg to 2.2 kg) (Sartorius, catalog number: MSE2203P-000-DR )
  26. Ice machine (multiple vendors)
  27. Desiccator (multiple vendors)
  28. Vacuum pump with manometer, condensation trap and tubing to connect desiccator to vacuum pump (multiple vendors)
  29. Fume hood
    Note: All manipulations involving the NIBF buffer must be performed inside a fume hood. Follow the safety regulations of your laboratory during the manipulations and for the waste disposal.
  30. Heraeus Multifuge 4KR centrifuge for 50 ml tubes (3,000 x g required) (Thermo Fisher Scientific, model: HeraeusTM Multifuge 4KR , catalog number: 75004461)
  31. Heraeus Fresco 21 centrifuge for Eppendorf tubes (16,000 x g required) (Thermo Fisher Scientific, model: HeraeusTM FrescoTM 21 , catalog number: 75002426)
  32. Epifluorescence microscope BX61 (Olympus) equipped with a cooled CCD camera (Hamamatsu Orca ER) (see Note 1) (Olympus, model: BX61 )
  33. Incubator cabinet (37 °C) (Memmert, model: Model 600 , catalog number: D06062)
  34. Rocking platform (Heidolph Instruments, model: Titramax 1000, catalog number: 544-12200-00 )
  35. Eppendorf ThermoMixer C (Eppendorf, model: ThermoMixer® C , catalog number: 5382000015) with a Smartblock for 1.5 ml tubes (Eppendorf, catalog number: 5360000038 )
  36. 1 L beaker glass (multiple vendors)
  37. Magnetic stir bar and magnetic stirrer (multiple vendors)
  38. Tube rotator (NeoLab, catalog number: 7-0045 )
  39. Incubator cabinet (16 °C) (GFL-Gesellschaft für Labortechnik, catalog number: 3032 ) placed in a cold room at 4 °C
  40. Qubit 2.0 (or Qubit 4) fluorometer with assay tubes, dsDNA HS Assay and dsDNA BR Assay (Thermo Fisher Scientific, model: Qubit 4, catalog number: Q33227 )
  41. Thermocycler (multiple vendors)
  42. Microwave (multiple vendors)
  43. Agarose gel electrophoresis equipment and accessories [microwave, tray (15 x 15 cm), combs, power supply, electrophoresis buffer, agarose, etc.] (multiple vendors)
  44. Covaris S220 AFA Ultrasonicator (Covaris) and associated equipment [snap-cap microTUBEs (with AFA-fiber and pre-split septum), chiller, software, computer (see Note 1)]
  45. Dark Reader blue light transilluminator (Clare Chemical Research, catalog number: DR46B )
  46. Agilent 2100 Electrophoresis Bioanalyzer or Agilent 4200 TapeStation System (Agilent Technologies, model: Agilent 2100 ) including accessories and consumables (see Note 1)
  47. GenPure Pro UV/UF (Thermo Fisher Scientific, catalog number: 50131950 )

Software

Software and computer hardware:
To analyze Hi-C/TCC sequence data, a computer server running a Unix operating system (e.g., Linux, Solaris, MacOS; see Note 1) or access to a cloud-computing system (e.g., CyVerse, http://www.cyverse.org) is required. Common UNIX command line tools (such as) need to be available. To accelerate CPU-intensive steps such as read alignment, access to a multi-core machine (> 16 CPU cores) is recommended. Depending on the number of samples to be analyzed, hard disk storage space needs to be allocated.
The following bioinformatics software need to be installed to carry out the primary data analysis described below:

  1. Casava, http://support.illumina.com/sequencing/sequencing_software/casava.html
  2. Cutadapt, http://cutadapt.readthedocs.io
  3. BWA-MEM, https://github.com/lh3/bwa
  4. SAMtools, http://www.htslib.org/download/
  5. BEDtools, http://bedtools.readthedocs.io

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Himmelbach, A., Walde, I., Mascher, M. and Stein, N. (2018). Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly. Bio-protocol 8(15): e2955. DOI: 10.21769/BioProtoc.2955.

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分类

植物科学 > 植物分子生物学 > DNA > DNA 测序

分子生物学 > DNA > DNA 结构

系统生物学 > 表观基因组学 > 染色质结构

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