title: GSM8736213: Th1_cells, BTP2 Treatment, Day4, biorep1 [Control-2_BTP2_Th1_S5]; Mus musculus; RNA-Seq
library_construction_protocol: ~3 million control and BTP2 Treated Th1 and Pathogenic  Th17 cells were harvested in TriZol reagent (Invitrogen) for RNA preparation.  3 Biological replicates per genotype were pooled after harvesting and divided into 3 technical replicates per condition. RNA was isolated using RNA purification kit (ThermoFisher Scientific), quantified and used for purification of mRNA, cDNA synthesis and library preparation using  KAPA stranded RNA seq-kit Sample following manufacturer's instructions. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. The workflow consists of depletion of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase and RNA fragmentaion. First strand cDNA synthesis using random priming followed by second strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (dscDNA), and incorporates dUTP into the second cDNA strand. cDNA generation is followed by A-tailing, adaptor ligation and PCR amplification. Different adapters were used for multiplexing samples in one lane. Sequencing was performed on Illumina HiSeq3000 for a SE 1x50 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq v2.19.1.403 software.