GSM1169473: ttTh2_2w_r2; Mus musculus; RNA-Seq
For sequencing total RNA: the total RNAs were purified using Qiagen's miRNeasy micro kit (Qiagen cat# 217084) plus Qiagen’s DNase set (Qiagen cat# 79254) for on-column DNase digestion option. For sequencing PolyA RNA: PolyA-tailed RNAs were purified from pure total RNA using Dynabeads® mRNA DIRECT™ Kit (invitrogen cat# 610.12). Both were performed according to the kits' user manuals. For STAT4 and STAT6 KO: total RNAs were isolated using mirVana (Applied Biosystems) isolation kit. RNA concentration was determined using the Qubit RNA broad range assay in the Qubit Fluorometer (Invitrogen) and RNA integrity was determined using Eukaryote Total RNA Nano Series II ChIP on a 2100 Bioanalyzer (Agilent). Three independent biological replicates were pooled for RNA-seq. For total RNA-seq library prep: 10ng of purified total RNA was used for cDNA amplification using the Ovation RNA-Seq System V2 (NuGEN Technologies, Inc. cat# 7102-08). then 200ng of ds cDNA was sonicated in Diagenode's Bioruptor (level M, for a  total of 30 minutes of 20 seconds ON and 20 seconds OFF) to a size range of 100-400bp. For mRNA-seq library prep:  a random primer (invitrogen cat# 48190-011) was used to prime the purified PolyA tailed-RNA and the cDNA synthesis was performed using SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen cat# 11917-010)  according to the user’s manual. The ds cDNA was subject to sonication in Diagenode's Bioruptor (level M, total of 30 minutes of 20 seconds ON and 20 seconds OFF). Indexed library was prepared using Illumina’s multiplexing sample prep oligonucleotide kit (Ref# 1005709) with Epicentre's End-It DNA End-repair Kit (Epcentre, cat# ER81050) according to the user's manual and Illumina's multiplexing sample preparation guide. For STAT4 and STAT6 KO: RNA-seq libraries were prepared from 4 ug of total RNA using the TruSeq RNA sample prep kit following manufacturer’s protocol (Illumina). Briefly, oligo-dT purified mRNA was fragmented and subjected to first and second strand cDNA synthesis. cDNA fragments were blunt-ended, ligated to Illumina adaptors, and PCR amplified to enrich for the fragments ligated to adaptors.