GSM8745412: pTh17_cells, ORAI 1/2/3 TKO, 48h, biorep1; Mus musculus; RNA-Seq
~3 million control and ORAI 2/3 DKO and ORAI 1/2/3 TKO 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 mRNA-Seq Kit. The workflow consists of mRNA enrichment and fragmentation, 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 end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq 3000 for a SE 1x50 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq v2.19.1.403 software.