title: GSM6659037: LentiMPRA transfected NPC cells DNA-seq replicate 3; Homo sapiens; OTHER
library_construction_protocol: DNA and RNA were extracted using AllPrep DNA/RNA mini kit (Qiagen) RNA was treated with Turbo DNase (Thermo Fisher Scientific) to remove contaminating DNA, and reverse-transcribed with SuperScript II (Invitrogen, 18064022) using barcodes specific primer (P7-pLSmp-assUMI-gfp), which has a unique molecular identifier (UMI - 10bp index). Barcode DNA/cDNA from each replicate were amplified with 3-cycle PCR using specific primers (P7-pLSmp-assUMI-gfp and P5-pLSmP-5bc-i#) to add sample index and UMI. A second round of PCR was performed for 19 cycles using P5 and P7 primers (P5, P7). The fragments were purified and further sequenced with NextSeq 15PE with 10-cycle dual index reads, using custom primers (R1, pLSmP-ass-seq-ind1; R2 (index read1 for UMI), pLSmP-UMI-seq; R3, pLSmP-bc-seq; R4 (index read2 for sample index), pLSmP-5bc-seqR2).