Bulk RNA-seq of BALB/c mice: Tgd17 from thymus - biological replicate 2
The RNA isolated from each sample was used to construct sequencing libraries with the SMART-Seq v4 Ultra Low Input RNA Kit for Illumina, following the manufacturer's protocol. First-strand cDNA synthesis (from total RNA or cells) is primed by the 3 SMART-Seq CDS Primer II A and uses the SMART-Seq v4 Oligonucleotide for template switching at the 5 end of the transcript. The first-strand cDNA selectively binds to SPRI beads leaving contaminants in solution which is removed by a magnetic separation. The beads are then directly used for PCR amplification. The Advantage 2 Polymerase Mix has been specially formulated for efficient and accurate amplification of cDNA templates by long-distance PCR. PCR-amplified cDNA is purified by immobilization on AMPure XP beads. The beads are then washed with 80% ethanol and cDNA is eluted with Elution Buffer. Prior to generating the final library for Illumina sequencing, the Covaris AFA system is used for controlled DNA shearing. The resulting DNA will be in the 200400 bp size range. These cDNA fragments then go through an end repair process, the addition of a single A base, and then ligation of the indexing adapters. The products are then purified and enriched with PCR to create the final cDNA library. The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified using the Agilent Technologies 4200 TapeStation (Agilent Technologies, Waldbronn, Germany).
SUB5836796
Tgd17_2_bulk_rseq