title: GSM8771445: pTh7_cells, Orai 1/2/3 TKO, 6 hrs, techincal rep 3; Mus musculus; RNA-Seq
library_construction_protocol: ~3 million control  and ORAI 1/2/3 TKO Pathogenic  Th17 cells were harvested in TriZol reagent (Invitrogen) for RNA preparation.  4 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 mRNA HyperPrep Kit. The workflow consists of depletion of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase. Next steps include 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 incorporating 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 NovaSeq6000 for a paired-end 2x50 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq v2.19.1.403 software.
identifiers.namespace: GEO
identifiers.id: GSM8771445_r1