1. Single cell capture and cDNA amplification
At the end of CRISPR screening, cells were prepared as single cell suspension and loaded into the Chromium Single Cell B Chip. The cDNA was generated, purified and amplified (the 1st PCR) following the manual of Chromium Single-Cell 3′ Reagent Kits v3 User Guide.
2.cDNA size selection
cDNA was separated into two parts according to the size.
(1) Long cDNA fragments, which mostly represented mRNA, were size-selected by 0.6x AMPure XP beads. The cDNA binding to the beads was called “long cDNA”, and will subject to mRNA library preparation. The "long cDNA" was then eluted in nuclease-free H2O following the 10x protocol.
(2) Short cDNA fragments, which mostly represented the index gRNA, were collected from the supernatant of the 0.6x beads selection, and then followed by a 1.2x size selection. The cDNA binding to the beads was called “short cDNA”. The "short cDNA" was then eluted in 25 uL nuclease-free H2O.
3. 3’ Gene Expression Library construction
The “long cDNA” was used for mRNA library preparation by following the manual of Chromium Single-Cell 3′ Reagent Kits v3 User Guide.
4. Index gRNA Library construction
The “short cDNA” was used for the index gRNA library preparation by nested PCR.
4.1. Index gRNA sequence enrichment
(1) In total of eight index gRNA enrichment PCR (the 2nd PCR) reactions were conducted, with each reaction including 3 uL template, 25 uL NEBNext Ultra II Q5 Master Mix (NEB #M0544S), 2.5 uL tRNA_Read2 primer (10uM), 2.5 uL P5_read1 primer (10uM) and nuclease free water up to 50 uL.
(2) The PCR program was set as: (1) 98 °C for 30 s, (2) 14 cycles of 98 °C for 10 s, 60 °C for 10 s, then 72 for 10 s and (3) 72 °C for 2 min.
(3) The PCR products were combined and purified by 0.7–1.0× double-sided size selection (collect the supernatant from the 0.7x beads size selection, and followed by 1.0x beads selection, then collect elution from the beads) and eluted in 80μl of nuclease free water.
4.2. gRNA library construction
(1) In total of five library preparation PCR (the 3
rd PCR) was conducted, with each reaction including 10 uL template, 25 uL NEBNext Ultra II Q5 Master Mix, 2.5 uL P7_Read2_Index2 primer (10μM) and 2.5 uL P5_read1 primer (10μM).
(2) The PCR program was set as: (1) 98 °C for 30 s, (2) 5 cycles of 98 °C for 10 s, then 54 °C for 15 s, then 65°C for 20 s and (3) 72 °C for 2 min.
(3) The PCR product was cleaned up and size selected with 0.7–1.0x AMPure XP beads.
(4) All eluted DNA was combined, and quantified. An aliquot was sent for NGS sequencing using the Illumina platform.