CRISPR Screen DropSeq Sequencing Library Prep Protocol
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DropSeq gRNA Library Prep Protocol
gRNA fragment sizes:
- Post SMART PCR: 182 bp
- Post Enrichment PCR: 239 bp
- Post Nextera PCR: 271 bp
- This protocol begins after the DropSeq SMART PCR. Split the PCR product into 45 uL (cDNA) and 5 uL (gRNA) aliquots. Add 30 uL Ampure XP beads to the 45 uL aliquot, and 10 uL Ampure XP beads to the 5 uL aliquot and do standard beads purification. Elute both aliquots in 15 uL water.
- Quantify the cDNA aliquot with Qubit (ideal range is between 0.2 and 1 ng/uL cDNA)
- Prepare gRNA enrichment PCR (15 uL gRNA purification product + 7 uL water + 1.5 uL DropSeq_gRNA_F + 1.5 uL P5-TSO-Hybrid + 25 uL 2X Kapa Hifi mix). Run for 20-24 cycles with standard Kapa Hifi protocol.
- Purifiy gRNA enrichment PCR product using gel purification (product should be 239 bp). Quantify with Qubit.
- Pool cDNA samples at a final concentration of 0.2 - 0.6 ng/uL, and follow the DropSeq Nextera protocol using the pooled sample
- Pool gRNA samples at a final concentration of 1-5 ng/uL. Load 5 ng gRNA for Nextera PCR (5 ng gRNA enrichment product + 1.5 uL N70X primer + 1.5 uL P5-TSO Hybrid) and run for 4-6 cycles. Run 5 uL on a gel and then purify with by adding #50 uL Ampure Beads (1.0x ratio) and follow standard purification protocol. Make sure you use different N70X indexes for the cDNA and gRNA libraries.
- Sequence cDNA and gRNA libraries with a Paired End 30 + 8 + 120 bp run. Aim for 20,000 cDNA reads per cell, and 2000 gRNA reads per cell.