GSM8734444: WT1; Rattus norvegicus; RNA-Seq
Total RNA was isolated using TRIzol. Isolated RNA was checked for integrity on an Agilent Bioanalyzer 2100; samples with RNA integrity number (RIN) >7.0 were used for subsequent processing. Total RNA was subjected to two rounds of poly(A) selection using oligo-d(T)25 magnetic beads (New England Biolabs). A paired-end (strand specific) cDNA library was prepared using the NEB Next Ultra-directional RNA-seq protocol. Briefly, poly(A)+ RNA was fragmented by heating at 94C for 10 min followed by reverse transcription and second strand cDNA synthesis using the reagents provided in the NEB Next kit. End-repaired cDNA was then ligated with double stranded DNA adapters, followed by purification of ligated DNA with AmpureXP beads. cDNA was then amplified by PCR for 15 cycles with a universal forward primer and a reverse primer with bar code. The sequencing of the cDNA libraries was performed on the Illumina NextSeq 500 platform (Illumina, San Diego, CA) using the single-read 1x75 cycles configuration.