GSM9439351: thymus_gex; Mus musculus; RNA-Seq
Postnatal day 6 thymic tissues were minced into ~1 mm pieces and digested with a mixture of papain (0.5 mg/mL), collagenase IV (0.25 mg/mL), and DNase I (0.1 mg/mL) at 37°C for 30 minutes. Released cells were collected with fresh enzyme mixtures added after 15 minutes. CD45+ hematopoietic cells were depleted using CD45 MicroBeads and LS columns (Miltenyi Biotec). CD45– stromal cells were stained with APC-conjugated anti-EpCAM antibody (clone G8.8) and purified by fluorescence-activated cell sorting (FACS). CD45–EpCAM+ TECs from three DKO and three control 6-day-old pups were independently labeled using TotalSeq™-A anti-mouse hashtag antibodies (BioLegend). Equal numbers of cells from each sample were pooled prior to encapsulation to minimize batch effects, targeting recovery of ~10,000 cells. An additive hashtag oligo (HTO) primer (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) was included in the cDNA amplification to enable efficient hashtag library preparation. Following cDNA cleanup, an HTO-specific library was generated by amplification with primers targeting Nextera Read 1 and Read 2. Libraries were pooled and sequenced using paired-end 2×150 bp reads, with a 10% PhiX spike-in, targeting a depth of 40,000 reads per cell for the cDNA library and 500 reads per cell for the HTO library.
GEO
GSM9439351_r1