GSM4272749: ESCCs, shCtrl-M-377; Mus musculus; RNA-Seq
Prior to cell isolation, all tools and solutions were autoclaved or filter sterilized. Mouse lungs were harvested and placed in cold DPBS, and were physically dissociated with scissors. To digest, dissociated tissues were mixed with 50ml of 1 mg/ml solution of collagenase type V (Sigma Aldrich, 9263), dissolved in prewarmed KSFM, and the mixture was stirred with a magnet for 20 min. at 37 oC. After digestion, the mixture was centrifuged twice at 1000 rpm for 5 min., resuspended in KSFM and plated on 10 cm cell culture dish. Cultured cells were washed with DPBS and replaced with new media every other day. After expansion, cells were trypsinized and flow cytometry sorted for YFP, and followed by western blotting to confirm p53 expression.  Total RNA was extracted with the RNAqueous kit (Invitrogen, AM1912). cDNA was synthesized using Takara SmartSeq v4 reagnets from 1ng of total RNA. cDNA was fragmented by Covaris AFA to ~150bp and adapters ligated using Rubicon Thrulplex DNAseq library prep kit from 2ng of cDNA according to manufacturer's protocol.
GEO Accession
GSM4272749