GSM1527728: Th2 d13 reactivated total RNA rep1; Homo sapiens; RNA-Seq
Total RNA naïve CD4+ cells and day 13 resting and restimulated Th1 and Th2 cells was obtained from samples previously used for microarray analysis. Poly-adenylated RNA was purified from 1 mg of total RNA using the Qiagen Oliogtex kit and ribosomal RNA was depleted from total RNA using Ribo-Zero Gold (EpiCentre), as per manufacturers instructions. 5' cap structures were removed with tobacco acid pyrophosphatase (TAP, Life Technologies) and RNA fragmented with potassium acetate (100mM) and magnesium acetate (30mM) at 94oC for 3 mins. RNA was then repaired with Antartic phosphatase and PNK, according to the instructions in the Illumina Directional mRNA-Seq Sample Prep Guide. Libraries were generated using the NEBNext Multiplex Small RNA Library Prep Set for Illumina kit, with 14 cycles of PCR amplification, and products between 130bp and 350bp gel purified. Libraries were quantified by qPCR (Library Quantification Kit, Kapa Biosystems) and average fragment size determined on an Agilent 2100 Bioanalyzer using DNA HS assays to pool libraries at equimolar concentrations.
GEO Accession
GSM1527728