GSM4409564: AK7986-6C; Mus musculus; RNA-Seq
E18.5 lungs were dissected without aeration of the lung. Lung lobes were snapfrozen in liquid nitrogen and RNA was harvested using Trizol reagent. mRNA was isolated from 1 ug total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer's instructions. Samples were eluted in 11.5ul of a mixture of NEBNext First Strand Synthesis Buffer and Random Primers, and 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) adding the beads solution in a ratio of 1:0.9. Dual unique indexing was done during the following PCR enrichment (8 cycles, 65 °C) 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 75bp single read on an Illumina NextSeq 500.
GEO Accession
GSM4409564