GSM4399743: WT-8wk-female; Mus musculus; RNA-Seq
Single cell pituitary suspensions were prepared by non-enzymatic methods as previously descripted (Ho et al., 2011) with minor modifications to adapt to the Drop-seq protocol (Macosko et al., 2015). Briefly, the pituitaries were isolated and washed with cold PBS, incubated with 1 ml of enzyme-free cell dissociation buffer (Life technologies, Carlsbad, CA) for 1 minute, passed through a 40 μm cell strainer, and then re-suspend in 10 ml of PBS. For Drop-Seq, single cell, polyA-containing barcoded beads (ChemGenes), and droplet generation oil (Bio-Rad) were each flowed into individual input ports on a microfluidic device (uFluidix). Cells in droplets were lysed and hybridized to beads. Beads were isolated and subjected to reverse transcription (Thermo Fisher) and exonuclease I (NEB) treatment, followed by PCR of full length transcripts (Kapa). Final libraries were created by tagmentation of cDNA (Illumina), and amplified by PCR and purified by SPRI beads. For 10x Genomics, single cell suspension of mouse pituitary was loaded onto a well on a 10x Chromium Single Cell instrument (10x Genomics). Library preparation was performed according to the manufacturer's instructions (Chromium Single Cell 3' Library & Gel Bead Kit v2).
GEO
GSM4399743