GSM8322720: MPRA library, CSTF3 gB, replicate 1; Homo sapiens; OTHER
48 hours post-transfection (7 days after original transduction with gRNAs), RNA was extracted using the Zymo DirectZol RNA purification kit with DNase I treatment following the manufacturer's protocol (Zymo R2052). mRNA was isolated from 5 µg total RNA per sample using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490S). mRNAs were reverse transcribed with SuperScript IV Reverse Transcriptase (Thermo Fisher 18090200) to generate cDNA following manufacturer instructions. The anchored poly dT primer for reverse transcription contained a Truseq Read 1 handle, an 8 bp sample barcode, and a unique molecule identifier (UMI) (TruseqR1-barcode-UMI-T18VN, see Supplementary Table 5 for primer sequences). After reverse transcription, RNA hydrolysis was performed using RNase H (Thermo Fisher 18021071) and then samples were purified using a DNA Clean & Concentrator-5 kit (Zymo D4013). Purified cDNA was amplified with KAPA HiFi HotStart ReadyMix (Kapa Biosystems 07958935001) using a forward primer containing additional Illumina adaptor sequences (P5-TruseqR1, 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC*T*C-3') and reverse primer containing a sample-specific barcode (P7-index-NexteraR2) (). Amplification was monitored with qPCR so that the reaction could be stopped early to minimize PCR bias with the following cycling conditions: 98 °C for 3 min; 15 cycles of 98 °C for 20 s, 60°C for 20 s and 72°C for 1 min; followed by 72°C for 5 min. After amplification, samples were 1X SPRI purified. The MPRA library was sequenced using an Illumina NextSeq Mid-Output (300 cycle) kit: allocating 16 cycles to Read 1, and 200 cycles to Read 2.