GSM8322691: NT, JKAMP,  replicate 2; Homo sapiens; OTHER
7 days post-transduction, RNA was extracted using the Zymo DirectZol RNA purification kit with DNase I treatment following the manufacturer's protocol (Zymo R2052). We divided RNA into 1ug aliquots, using a separate aliquot for each targeted locus. For each locus, the 1 µg of total RNA was 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 Nextera Read 2 handle (NexteraR2-T18VN, 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTTTTTTTTTTTTTTTTVN-3'). 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). In the first locus-specific PCR, purified cDNA was amplified with KAPA HiFi HotStart ReadyMix (Kapa Biosystems 07958935001) using the locus-specific primer (Figure S1) and adds a Truseq Read 1 handle and a reverse primer containing a sample-specific barcode (P7-index-NexteraR2) (Supplementary Table 3) with the following cycling conditions: 98 °C for 3 min; 15 cycles of 98 °C for 20 s, 65°C for 20 s and 72°C for 1 min 20 s; followed by 72°C for 5 min. After amplification, samples were 1X SPRI purified. In the second PCR to prepare samples for next-generation sequencing, the purified product from the previous PCR was amplified with KAPA HiFi HotStart ReadyMix (Kapa Biosystems 07958935001) using a forward PCR primer add sequencing adaptors (P5-TruseqR1, 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC*T*C-3') and a reverse P7 PCR primer (5'-CAAGCAGAAGACGGCATACGAGAT-3') 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 20 s; followed by 72°C for 5 min. Samples were sequenced using an Illumina MiSeq (300 cycle kit), allocating 200 cycles for R1.