GSM8012093: Human Sertoli cells, therapeutic buffer treatment, 48h, MOI 10, replicate 1; Homo sapiens; RNA-Seq
Cells were harvested 24h or 48h post-infection, using a cell scraper. Harvested cells were centrifuged to remove media, and were immediately snap frozen. RNA extraction was carried out using the RNeasy lipid tissue kit (Qiagen GmBH, Hilden, Germany) according to the manufacturer's instructions. On-column DNAse treatment was performed using DNAse I reagent. Total RNA samples were assessed by Qubit and Bioanalyzer. Samples with a RIN over 7 were selected for RNA-seq. Libraries were generated using an in-house multiplex RNA-seq method (version 01/09/2021) and were prepared using 25 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. Illumina P5 was added by tagmentation by Nextera transposase and PCR and Illumina P7 was added by PCR. The library was designed so that the forward read (R1) used the standard R1 primer to sequence the cDNA in the sense direction for transcript identification. The index read (I1) used the standard i7 primer to sequence directly into the sample index and then the 10 bp UMI. Samples were prepared as two libraries, so an additional index read (I2) sequenced the i5 library index. Sequencing (R1 61 bp; I1 18 bp; I2 8 bp) was performed on a NextSeq 2000 (Illumina) P3 50 cycle run, as per the manufacturer's instructions. On-board denaturing and clustering were done using 900 pM of library pool (Illumina Protocol 1000000109376 v3 Nov2020).