BrdU-seq

Day 1

1. Cell seeding 

• Seed U2OS cells in 10 cm dish
• Seed 1 Mio cells per dish
• Seed 3 biological replicates per condition

Day 2

1. Cell synchronization and treatment of cells 

NOTE: Timing are stated as an example for handling 3 biological replicates

16.01.25 Block 1st round (15:00, 15:20, 15:40)

• Remove culturing medium
• Add fresh medium supplemented with thymidine to a final concentration of 2 mM (1:50)
• Culture cells in a tissue culture incubator at 37 °C for 18 h

17.01.24 Release (9:00, 9:20, 9:40)

• Remove thymidine by washing cells through addition of 10 mL pre-warmed 1x PBS 
• Shake gently and let stand for 1 min to wash, discard PBS
• Add 10 mL of pre-warmed fresh medium and incubate for 9 h in a tissue culture incubator at 37 °C

17.01.24 Block 2nd round (18:00, 18:20, 18:40)

• Add second round of thymidine to a final concentration of 2 mM
• Culture cells at the tissue culture incubator for another 18 h at 37 °C

18.01.24 Final release (2h into S-phase) 

• R1 12:00 - Release into S-phase by thymidine removal
• R2 12:20 - Release into S-phase by thymidine removal
• R3 12:40 - Release into S-phase by thymidine removal 

• R1 2h - 13:30 - Add 50 µM BrdU to S-phase cells
• R2 2h - 13:50 - Add 50 µM BrdU to S-phase cells
• R3 2h - 14:10 - Add 50 µM BrdU to S-phase cells

• R1 2h - 14:00 Harvest 2h cells
• R2 2h - 14:20 Harvest 2h cells
• R3 2h - 14:40 Harvest 2h cells

• Resuspend cells in medium
• Pellet cells for 2 min at 1500 rpm at 4°C
• Wash cells with PBS and pellet again for 2 min at 1500 rpm at 4°C
• Remove supernatant and resuspend in 900 µL PBS
• Distribute 450 µL each into two new 1.5 mL low-binding tube
• Spin down again 2 min 400g at 4°C
• Remove supernatant and snap freeze in liquid nitrogen
• Store at -80°C until further use

Day 3

1. DNA purification

• Resuspend cell pellet in 100 µL TE buffer and add 100 µL IRN buffer, 0.5% SDS and 10 µL Proteinase K (10 mg/mL). 
• Incubate @ 37°C overnight. 

Day 4

• Add 200ul phenol:chloroform:isoamyl alcohol (25:24:1, v/v) 
• Vortex sample 2x for 20s. Sample will turn white/milky.
• Spin 10 min @ 15000 rpm (max. speed) for phase separation.
• Transfer the upper aqueous phase (which contains the DNA) into a new tube.
• Add 5 µL RNase A (10 mg/mL) and incubate for 1.5h at 37°C.
• Extract DNA with 200ul chloroform (Vortex sample 2x for 20s. Sample will turn white/milky. 
• Spin 10 min @ 15000 rpm (max. speed) for phase separation.
• Transfer 180ul of the upper aqueous phase into new tube.
• Add 1/10th volume of NaAc pH5.2 (20 µL) and 2.5 volumes of 100% ethanol (0.5 mL), add 1ul 10 mg/ml glycogen to the sample to improve      precipitation
• Incubate at -20°C for at least 1h or ON

• Spin samples @ 13000 rpm at 4°C for >30 min.
• Wash 1 mL with 70% EtOH
• Spin samples @ 13000 rpm at 4°C for 10 min.
• Resuspend pellet with 50 µL H2O
• Merge technical purification replicates with each other and quantify with Qbit dsDNA BR Kit
• Dilute 20 µg of DNA in 135 µL of H2O

Day 5

• 16:00 - Prepare 10 µL Dynabeads Protein G by incubating with 2 μg anti-BrdU antibody at 4°C overnight with rotation.  
• 16:05 - Wash beads 3x with PBS-T 0.02%
• 16:15 - Add 2 µg (80 µL of 0.025 µg/µL) BrdU antibody (AB023)

1. Covaris Sonication and BrdU DNA immunoprecipitation

• Use AFA microTube with Snap Cap (130µL)
• Start with 20 µg total input material
• Fill up to 130 µL with water

Covaris Settings

• Sonicate for 10 min per sample (2h total for 12 samples)

• 16:00 - Use water to increase the total volume to 200 µL
• 16:10 - Centrifuge sonicated DNA at 3000 rpm at 4°C for 5 min, store at 4°C ON

Day 6

• Denature DNA at 100°C for 10 min, put on ice immediately afterwards
• Set aside 15 µL of DNA as an input 
• Mix input DNA with 35 µL of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS)
• Mix 170 µL of DNA with 180 µL of 2X blocking solution (2% BSA, 2XPBS, 0.2% Tween20), 10 µL Dynabeads Protein G (Thermo Fisher Scientific) and 2 μg anti-BrdU antibody (AB023) in DNA low binding tubes, incubated at 4°C overnight with rotation.

Day 7

• Wash BrdU-labeled DNA bound to beads twice with 700 µL of lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na Deoxycholate)
• Wash twice with 700 µL of lysis buffer 2 (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na Deoxycholate), 
• Wash twice with 700 µL of wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl,1 mM EDTA, 0.5% Na Deoxycholate, 0.5% Igepal CA-630). 
• Resuspend beads in 700 µL of TE and centrifuged for 3 min, 2000 rpm at 4°C
• Remove supernatant and resuspend beads in 100 µL elution buffer with 0.5 mg/µL Proteinase K and incubate at 65°C for 10 min at 400 rpm
• Transfer supernatant to new low binding tube
• Repeat previous elution step and merge the supernatant with the previously collected eluate (200 µL total eluate)
• Add 150 µL TE and final concentration of 0.5 mg/ml Proteinase K (Promega) to 50 µL Input DNA and incubated at 37°C for 1 h (200 µL total)
• Incubate samples at 37°C for 1h → Protease K digestion

1. DNA purification

Day 8

Subsequent steps done for all samples

• Add 1 µL of 1ng/µL p-BlueScript spike-in plasmid to all samples
• Extract DNA with 200 µL chloroform (Vortex sample 2x for 20s. Sample will turn white/milky. 
• Spin 10 min @ 15000rpm (max. speed) for phase separation.
• Transfer 180 µL of the upper aqueous phase into new tube.
• Add 1/10th volume of NaAc pH5.2 (20 µL) and 2.5 volumes of 100% ethanol (0.5 mL), add 2 µL 10mg/mL glycogen to the sample to improve precipitation.
• Incubate @ -20°C for 1h or ON

• Centrifuge at max speed (13000 rpm, cooled centrifuge) at 4 °C for at least 30 min 
• Remove supernatant
• Wash with 700 µL 70% EtOH
• Centrifuge at max speed (13000 rpm, cooled centrifuge) at 4 °C for 10 min 
• Let pellet air dry for a few minutes until visible EtOH is gone 
• Resuspend pellet with 30 µL H2O
• Store at 4 °C ON

Day 9

6. qPCR

• Dilute samples 1:3 with water (dilute 10 µL with 20 µL with H2O)