GSM1904556: WT sample 2; Mus musculus; RNA-Seq
Postnatal day 3 or 4 Scnn1a-Cre; RosamTmG/+ and Scnn1a-Cre; RosaSox2/+ littermate lungs were dissected in PBS. Around 3 mm wide strips were cut along the lobe edge, minced into pieces with forceps and digested in PBS with 2 mg/mL collagenase type I (Worthington,16 CLS-1) and 0.2 mg/mL DNase I (Worthington, D) for 40 min at 37 °C. An equal volume of 0.25% Trypsin/EDTA (Invitrogen, 25200-056) was added and incubated for another 10 min at 37 °C. Fetal bovine serum (FBS, Invitrogen, 10082-139) was added to a final concentration of 5% and the tissue was triturated for 40~50 times. Dissociated cells were spun down at 6000 rpm for 1 min, resuspended in red blood cell lysis buffer (15 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA, pH 8.0) for 3 min, washed with PBS with 1% FBS, SYTOX-red (Invitrogen, S34859) added to exclude dead cells and sorted on a BD Biosciences Influx sorter. RNA was extracted from at least 105 purified cells using Trizol reagents (Invitrogen, 15596018) and an RNeasy Micro kit (Qiagen, 74004). 100~200 ng total RNA was used to prepare an RNAseq library using an mRNA isolation kit (New England BioLabs, E7490) and a NEBNext Ultra RNA library prep kit (New England BioLabs, E7530S) with a final double (0.65 x -1 x bead volume) size selection step using a SPRIselect reagent kit (Beckman Coulter, B23318). Sequenced on an Illumina HiSeq2000.
GEO Accession
GSM1904556