title: GSM4141870: Bgl-C-T; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow was isolated and single cell suspensions were prepared. GMP were FACS-sorted (Lin—c-Kit+Sca1—CD16/32+CD34+) and RNA was isolated using RNeasy Plus Microkit (Qiagen). mRNA was isolated from 50 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instructions. Final elution was done in 15ul 2x first strand cDNA synthesis buffer (NEBnext, NEB). Samples were then directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep II, NEB). For ligation custom adaptors were used 1: (Adaptor-Oligo 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 ligaton adapters were depleted by an XP bead purification (Beckman Coulter) adding the beads solution in a ratio of 1:1. Indexing was done during the following PCR enrichment (14 cycles, 65 °C) using custom amplification primers carring the i7 index sequence indicated with 'NNNNNNNN'. (Primer1: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, primer2: 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 beads purifications (1:1) libraries were quantified using Qubit dsDNA HS Assay Kit (Invitrogen).