GSM2943024
GSM2943024: 1_L28_C68; Mus musculus; RNA-Seq
To obtain mesenchymal cells from the adult lung, the pulmonary vasculature was cleared and lungs were gently inflated with digestion buffer prior to isolation and mincing of the lung. All tissue was digested at 37C for 45 min using Elastase and DNase I, triturated and subsequently filtered using 100µm and 40µm strainers to obtain a single cell suspension. Mesenchymal cells were enriched by magnetic-assisted cell sorting using CD45, Pecam, and EpCAM depletion. Cells were loaded into the Fluidigm C1 microfluidic platform, where single cells were captured. Lysis of single cells, reverse-transcription of mRNA into cDNA as well as preamplification of cDNA occured within the microfluidic device using reagents provided by Fluidigm as well as the SMARTer Ultra Low RNA kit for Illumina (Clontech). External RNA spike-in transcripts (ERCC spike-in Mix, Ambion) were added to all single cell lysis reactions. As a bulk control, cDNA was prepared from approximately 200 cells in a PCR tube using the same protocol and reagents as used for the single cells. Libraries were prepared using Illumina Nextera XT kit per illumina's protocols.
GEO
GSM2943024
GEO Accession
GSM2943024