GSM5598005: CD20- T cells [S20]; Homo sapiens; RNA-Seq
CD20+ and CD20- CD3+ CD8+ CD45RO+ CCR7- lymphocytes were sorted in 96-wellsplate containing lysis buffer 2 µl lysis buffer (0,2% Triton X-100, 2U RNase inhibitor) supplemented with 1 µl 10 µM oligo-dT primer and 1 µl 10 mM dNTP mix. After sorting, the plate was spun down, incubated at 72°C for 3 minutes and stored on ice. A modified SMARTseq2 protocol using custom-made primers was used. In brief, SmartScribe reverse transcriptase (Westburg-Clontech) and a template switching oligo (BC-TSO) were used to generate cDNA. Next, a PCR preamplification step was done with KAPA HiFi HotStart Ready Mix (Roche Diagnostics) and a custom-made PCR primer. The cDNA samples were purified using Ampure XP beads (Beckman Coulter) in a ratio of 0,6:1 (Ampure bead : cDNA). Samples were analyzed on a 2100 Bioanalyzer using a PerkinElmer LabChip GX high-sensitivity DNA chip (Agilent) and on a Qubit™ 4 Fluorometer (ThermoFisher Scientific) according to manufacturer's instructions. Next, 500 pg of each sample was tagmented and N7xx and S5xx index adapters were used for barcoding according to the Illumina Nextera XT DNA sample preparation kit (Illumina). Thereafter, samples were purified using Ampure XP beads (ratio 0,6 Ampure : 1 cDNA) and analyzed on a 2100 Bioanalyzer. Samples were equimolar pooled (4 nM) and subsequently sequened on an Illumina Nextseq500 2500 using 75 bp single-end reads.
GEO Accession
GSM5598005