GSM5583254: C20.5; Mus musculus; RNA-Seq
Murine lungs were harvested immediately after the time of sacrifice, sliced into small pieces, homogenized using BeadBlaster Microtube Homogenizer (90sec, 4000rpm) in the presence of QIAzol, and used for total RNA extraction using the miRNeasy Mini Kit. For the preparation of RNA‐Seq libraries, total RNA was fragmented into average size of 300 nucleotides by chemical heat (95°C) treatment for 4:30 min (NEBNext Magnesium RNA Fragmentation Module). The 3′ polyadenylated fragments were enriched by selection on poly dT beads (Dynabeads Invitrogen). Strand‐specific cDNA was synthesized using a poly T‐VN oligo (18 T) and Affinity Script RT enzyme (Agilent). Double‐strand DNA was obtained using Second strand synthesis kit (NEB). DNA ends were repaired using T4 polynucleotide kinase and T4 polymerase (NEB‐Next). After the addition of an adenine base residue to the 5′ end using Klenow enzyme (NEB‐Next), a barcode Illumina compatible adaptor (IDT) was ligated to each fragment. The washed DNA fragment was amplified by PCR (12 cycles) using specific primers (IDT) to the ligated adaptors. The quality of each library was analyzed by TapeStation (Agilent).