title: GSM6969663: Ect1 cells, IFN lambda, R1; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were harevested in RLT buffer (Qiagen) with added β-mercaptoethanol. Lysates were homogenized by passing through a 20-gauge needle attached to a sterile plastic syringe. Total RNA was extracted from lysates using the Qiagen RNeasy kit, including removal of DNA contamination using the Qiagen RNase-free DNase set as per the manufacturer's instructions. Libraries were generated using an in-house multiplex RNA-seq method (MHTP, Medical Genomics Facility) and were prepared using 20 ng of total RNA input. An 8 bp sample index and a 10 bp unique molecular identifier (UMI) were added during initial poly(A) priming and pooled samples were amplified using a template switching oligonucleotide. The Illumina P5 and P7 sequences were added by PCR and Nextera transposase, respectively. The library was designed so that the forward read (R1) utilised a custom primer (5' GCC TGT CCG CGG AAG CAG TGG TAT CAA CGC AGA GTA C 3') to sequence directly into the index and then the 10 bp UMI. The reverse read (R2) used the standard R2 primer to sequence the cDNA in the sense direction for transcript identification. Paired-end sequencing (R1 19 bp; R2 72 bp) was performed on the NextSeq 550 (Illumina), using the v2.5 High Output Kit.