GSM9588876: Atg14_flox mice4, naive; Mus musculus; RNA-Seq
Lung cells were harvested PBS perfusion, placed in DMEM containing 10% FBS, minced finely, and digested at 37 ⁰C for an hour with mechanical disruption with a stir bar and enzymatic digestion with Liberase Blendzyme III (Roche), hyaluronidase (Sigma), and DNase I (Sigma).RNA was isolated from cells using the RNeasy minikit (QIAGEN) in accordance with the manufacturer's instructions. mRNA was extracted from cell lysates by means of oligo-dT beads (Invitrogen). For cDNA synthesis, we used custom oligo-dT primer with a barcode and adaptor-linker sequence (CCTACACGACGCTCTTCCGATCT-XXXXXXXX-T15). After first-strand synthesis, samples were pooled together based on Actb qPCR values and RNA-DNA hybrid was degraded with consecutive acid-alkali treatment. Then, a second sequencing linker (AGATCGGAAGAGCACACGTCTG) was ligated with T4 ligase (NEB) followed by SPRI (Solid Phase Reversible Immobilisation) clean-up. The mixture then was PCR enriched 12 cycles and SPRI purified to yield final strand-specific RNA-seq libraries.