alias: E-MTAB-7594:LCMV_1_p
title: Illumina HiSeq 2500 paired end sequencing; Gene expression comparing Memory Bystander T cell Function with and without LCMV infection
design: Gene expression comparing Memory Bystander T cell Function with and without LCMV infection
library_construction_protocol: CD8+ OT-1 T cells were sorted and resuspended and frozen in TRI Reagent® (ZymoResearch). RNA was isolated using the Zymo Research Direct-zolTM RNA MicroPrep (R2060) according to the manufacturer's protocol. RNA-seq libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit for Pico Input Mammalian from Clonetech. Briefly, total RNA samples (0.25–10 ng) were reverse-transcribed using random priming into double-stranded cDNA in the presence of a template switch oligo (TSO). PCR amplification using primers binding to these sequences can now be performed. The PCR adds full-length Illumina adapters, including the index for multiplexing. Ribosomal cDNA is cleaved by ZapR in the presence of the mammalian- specific R-Probes. Remaining fragments are enriched with a second round of PCR amplification using primers designed to match Illumina adapters.The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Tapestation (Agilent, Waldbronn, Germany). The product is a smear with an average fragment size of approximately 360 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.
identifiers.namespace: ETH Zuerich
identifiers.id: E-MTAB-7594:LCMV_1_p