GSM6069659: day03-lung; Mus musculus; RNA-Seq
Murine lung cells were isolated at 0, 3, 7,10, 14 and 21 days post-bleomycin administration. Lungs were cut into small pieces with a razor blade and digested enzymatically using 0.25 mg/ml Liberase TM (Roche) and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO) for 60 min at 37°C. Erythrocytes and dead cells were removed by 25%/65% Percoll (GE Healthcare, Buckinghamshire, UK) density separation. Resulting cells were stained with Calcein AM for 5 min at 37°C, immediately chilled on ice, and filtered through 30 μm filter (Miltenyi). 15000 cells were loaded onto BD Rhapsody cartrige and captured cell number was asessed by BD Rhapsody scanner. Cell lysis, mRNA trap, cDNA synthesis, and primer digestion were performed according to the manufacture's instruction. cDNA amplification was performed by TAS-Seq protocol (Shichino et al bioRxiv 2021). Sequencing libraries from amplified cDNAs were produced by using NEB UltraII FS Kit (New England Biolabs) with some modifications. Sequencing was performed by using a Novaseq S2 200 cycle kit and a Novaseq 6000 sequencer (Illumina) according to the manufacture's instructions. 20% PhiX spike-in was used for sequencing.