GSM9277813: Donor 4, Nonproductive infection; Homo sapiens; RNA-Seq
Cellular RNA and DNA from FACS-sorted CD4+ TSCM or total CD4+ T cells were isolated using Allprep DNA/RNA Micro kit (Qiagen). RNA was treated on-column with DNase I (Qiagen) and quantified using Qubit HS RNA assay kit. RNA quality was determined using Agilent RNA 6000 Nano Kit on Agilent Bioanalyzer 2100. Samples with RNA integrity number > 8 were used for library preparation. RNA-seq libraries were produced using the SMART-Seq mRNA LP (with UMIs) kit (Takara Bio), following manufacturer's instructions and starting from an input of 10 ng of total extracted RNA per sample. Along with sorted RNA samples, positive control (intact RNA of known concentration provided by the kit) and negative control (nuclease-free water, provided by the kit) were included in each reaction. RT-cDNA was amplified through 10 PCR cycles, and amplified cDNA was purified (AMPure XP Beads, Beckman Coulter) and run on Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit (Agilent). cDNA input was normalized at 2.5 ng before library prep reactions and fragmented libraries were PCR amplified through 15 PCR cycles. Amplified libraries were singularly purified (AMPure XP Beads, Beckman Coulter), run on Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit (Agilent) and quantified at Qubit using Qubit dsDNA BR Assay kit (Invitrogen). Individual libraries were normalized at 10 nM and pooled together. Molarity of the pooled library was confirmed through KAPA Library Quantification PCR (KAPA Biosystems). Pooled libraries were sequenced on a NovaSeqX Plus instrument (Illumina) using a paired-end sequencing approach with reads of 150 base pairs in length (PE150 strategy).