title: GSM8857144: hNSC, SuRE44-2, 24h, biol rep 1; Homo sapiens; OTHER
library_construction_protocol: 24 hours after transfection, cells were washed with 1x PBS, manually scraped and resuspended in 30 ml TRI Reagent (Sigma-Aldrich, T9424) and stored at -80°C. RNA was isolated using Trisure (#BIO-38032; Bioline). Briefly, total RNA was divided into 10-μl reactions containing 500 ng RNA and treated with 10 units DNase I for 30 min (#04716728001; Roche) and DNase I was inactivated by addition of 1 μl 25 mM EDTA and incubation at 70°C for 10 min. Next, cDNA was produced by first 1 μl of 10 μM gene-specific primer targeting the GFP ORF (247JvA) and 1 μl dNTP (10 mM each) and incubating for 5 min at 65°C. Then 4 μl of RT buffer, 20 units RNase inhibitor (#EO0381; ThermoFisher Scientific), 200 units of Maxima reverse transcriptase (#EP0743; ThermoFisher Scientific) and 2.5 μl water were added, and the reaction mix was incubated for 30 min at 50°C followed by heat inactivation at 85°C for 5 min. Per replicate, 20–30 reactions were performed in parallel. Each 20 μl reaction was then PCR amplified (1 min 96°C, 20 × (15 s 96°C, 15 s 60°C, 15 s 72°C)) in a 100-μl reaction with MyTaq Red Mix and primers 151AR (containing the S1 and p5 adaptor) and (index variants of) 211JvA (containing the S2, index and p7 adaptor) for 21 cycles. Reactions were then pooled and 500 μl was purified using a PCR purification kit (#BIO-52060; Bioline) and then size-selected in two rounds using CleanPCR beads (#CPCR-0050, CleanNA). Reverse-transcribed, PCR-amplified barcodes were subjected to single-read (SE) 50-bp high-throughput sequencing