title: GSM8800346: phNPC DNA ENIGMA MPRA (Stimulation) Rep12; Homo sapiens; OTHER
library_construction_protocol: At exactly 48 hours after CHIR or vehicle treatment, RNA and DNA was extracted from the cells using the Quick-DNA/RNA Miniprep Plus Kit (Zymo, cat#D7003), with 600 µL of Shield Buffer and 600 µL of Lysis Buffer per well. Extracted RNA was reverse-transcribed into cDNA using SuperScript IV Reverse Transcriptase (Invitrogen, cat. no. 18090050) and a primer designed to bind downstream of the barcodes (Lib_Hand_RT). The resulting cDNA was then amplified with Lib_Hand and Lib_seq_Luc_R primers, utilizing NEBNext 2X Q5 Hifi HS Mastermix, and then purified using the Zymo DNA Clean and Concentrator kit-25 (Zymo, cat. no. D4033). Additionally, 4 µL of DNA from each well was PCR amplified in four separate PCR reactions using Lib_Hand_RT and Lib_Seq_Luc_R primers with NEBNext 2X Q5 Hifi HS Mastermix, followed by purification with the Zymo Clean and Concentrator kit-25. P5 and P7 adapter sequences and i7 demultiplexing indices were then incorporated into both cDNA and DNA libraries through PCR using P5_seq_Luc_F and P7_Ind_#_Han primers with NEBNext 2X Q5 Hifi HS Mastermix. The final PCR products were purified and size-selected using AMPure XP Beads (Beckman Coulter, cat. no. A63881) at 0.7X and 0.9X ratios to isolate a 273 bp DNA fragment.