Barcode to gRNA assignment

Barcode to gRNA assignment (via PacBio Sequencing)

Introduction

This protocol details the steps to generate a PacBio sequencing library which is used to assign each random nucleotide barcode to its corresponding gRNA.

Materials

› Barcoded gRNA plasmid library

› I PacBio sequence the plasmid library before the divergent promoter insertion (see "Plasmid library construction" protocol). The same barcode to gRNA assignment can then be used for each plasmid library constructed by inserting the divergent promoter into the barcoded gRNA plasmid library.
 

› Set of restriction enzymes that cleave on either side of the dual-barcode and gRNA.

› For my library, I used HindIII, PmeI, BglII, and XhoI. These enzymes are compatible with dual barcoded gRNA libraries constructed from the parent vector "pNTI743 dual-barcoded gRNA parent vector" (available soon on addgene)
 

› Agarose Gel for DNA size selection and purification

› Zymoclean Gel DNA Recovery Kit (Zymo #D4001)

› Nanodrop for DNA concetration quantification

› SMRTbell Express Template Prep Kit 2.0 (#100-938-900)

› AMPure XP beads

› Tape Station/Bioanalyzer + necessary Tape reagents

Procedure

PacBio Library Preparation

1. Because PCR amplification can introduce artifacts and PCR-mediated exchange, barcoded gRNA DNA fragments are genereated by digesting the barcoded gRNA plasmid library. For libraries constructed in the pNTI743 (see addgene) backbone, digest 5μg of dual barcoded gRNA plasmid library with HindIII and PmeI according to manufacturer protocol. Separately digest 5μg of dual barcoded gRNA plasmid library with BglII and XhoI according to manufacturer protocol.
   During plasmid library construction, the dual barcode sequence was pre-digested with HindIII, PmeI, BglII, and XhoI to avoid dropout at this step in the PacBio libary prep. (See "Plasmid library construction" protocol). The gRNA library, however, still contains sequences that can be digested by these restriction enzymes. Two separate sets of restriction enzymes were thus used to ensure gRNA sequences could be recovered in at least one restriction digestion set.
2. The ~1200-bp fragment for each digestion, containing dual barcode and gRNA, is gel extracted according to Zymoclean Gel DNA Recovery Kit (Zymo #D4001) instructions. Elute in nuclease-free water.
3. The two purified DNA fragments are quantified by nanodrop and mixed equimolarly. Save a little of the purified fragments for downstream tapestation diagnostics. The equimolar mix is used as the input for the dumbell adapter ligation reaction. See accompanying modified protocol for using SMRTbell Express Template Prep Kit 2.0 (#100- 938-900), which has replaced the PCR-amplification strategy with the described plasmid digestion strategy for generating input DNA.
4. Use Ampure XP beads to remove unligated dumbell adapters from the PacBio library. Compare the final PacBio library against the gel-purified DNA fragments on a tape station/bioanalyzer to confirm efficient dumbell ligation. You should see a peak shift upwards in the sample containing the dumbel adapters. Sample is ready for sequencing using the Sequel II system. See PacBio documentation for more details about sequencing.

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