Primary T-cell, Patient 35
Approximately 500 thousand to 1 million cells were lyzed and stored in 700uL Qiazol lysis reagent (QIAGEN, ref: 79306). To isolate RNA, 140 uL of chloroform was added. After centrifugation at 12000 x g for 15 min, approximately 350 uL of the upper layer containing the RNA was transferred and mixed with 350 uL of 70% ethanol. RNA isolation was continued from this point using the RNeasy MinElute kit (QIAGEN, ref: 74204) protocol. RNA libraries were then generated by NovoGene UK using a custom protocol based on cDNA synthesis using random hexamer primers and polyA mRNA enrichment. Sequencing was performed on the NovaSeq6000 platform using NovaSeq S4 flow cells at a target depth of 40 million reads per library and generating 150bp paired-end reads.