GSM5551719: Lung Bmem 3.1; Mus musculus; RNA-Seq
Lungs and spleen tissue were digested in 1mg/ml Collagenase D and 10ug/ml DNase1 in RPMI for 45min. After digestion, Ficoll-paque gradient was also run to enrich for lymphocyte fraction before staining and sorting. 200 cells (per sample) were sorted directly into lysis buffer and frozen at -80 prior to library construction. Library construction was performed as previously described, using a Smart-seq2 protocol adapted to low cell numbers (Radtke 2019; PMID:30687317). 19 cycles of preamplification were used for all samples. cDNA purification was performed using Ampure XP beads (Beckman Coulter). Libraries were analyzed with a High Sensitivity Analyser (Agilent) and cDNA tagmentation was performed with the Nextera XT DNA Sample Preparation kit (Illumina). Libraries were quantified using PicoGreen (Illumina), sized using the High Sensitivity Analyser and equal amounts of tagmented cDNA from each library were pooled.