GSM6315021: RH2hKO r8 (s013); Mus musculus; RNA-Seq
Lineage-negative BM cells were prepared from RH2hKO mice and RH2hWT littermate controls (n=4/genotype) and 30,000 – 100,000 GMPs (LE-K CD16/32hiCD34+) were sorted. Total RNA was prepared using the RNeasy Micro Kit (Qiagen). RNA quality was analysed using a Agilent 2100 Bioanalyzer (Agilent Technologies) and samples were frozen at ‑80°C. mRNA was isolated from 37 – 50 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer's instructions. Samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation (Ultra II Directional RNA Library Prep, NEB). For ligation custom adaptors were used (Adaptor-Oligo 1: 5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3', Adaptor-Oligo 2: 5'-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3'). After ligation, adapters were depleted by an XP bead purification (Beckman Coulter). Unique dual indexing was done during the following PCR enrichment (15 cycles) using custom amplification primers carrying the same sequence for i7 and i5 index (Primer 1: AAT GAT ACG GCG ACC ACC GAG ATC TAC AC NNNNNNNN ACA TCT TTC CCT ACA CGA CGC TCT TCC GAT CT, Primer 2: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two more XP bead purifications (1:0.9), libraries were quantified using the Fragment Analyzer (Agilent). For Illumina flowcell production, samples were equimolarly pooled and sequenced on Illumina NovaSeq 6000 in 2 x 50 bp paired-end mode, resulting in at least 35 million fragments per sample.