title: GSM7864907: Two pooled e14.5 pituitary glands from C57BL6 strain; Mus musculus; RNA-Seq
library_construction_protocol: One e12.5 pituitary gland from a retinoic acid reporter mouse (RARE-LacZ JAX strain #008477) was dissociated in an enzyme mix containing 0.5% w/v collagenase type 2 (Lorne Laboratories, Reading, UK), 0.1× trypsin (Invitrogen, Carlsbad, CA), 50 μg/mL DNase I (Worthington Biochemical, Lakewood, NJ), and 2.5 μg/mL amphotericin B (Fungizone; Invitrogen) in Hanks balanced salt solution (Invitrogen) for 1 hour at 37°C.  For the e14.5, 2 C57BL/6J e14.5 samples were dissociated into single cells in 50 μl of a solution containing 1mg/ml Papain (Roche, cat#10108014001), 5mM L-Cysteine, 1mM EDTA, and 0.6mM 2-mercaptoethanol. Dissociation was performed at 37°C for 5 minutes, with titration every 2 minutes. Single-cell RNA libraries were prepared by the University of Michigan's Advanced Genomics Core under a standardized pipeline on the 10x Genomics Chromium platform and controller, following manufacturer's instructions for Single Cell 3' v3 reagents, and sequenced on the NovaSeq6000 platform with an S4 chip.  From our previous description in Cheung et al., 2018 Endocrinology: Following generation of single-cell gel bead-in-emulsions (GEMs), reverse transcription was performed and the resulting post-GEM reverse transcription product was cleaned up using DynaBeads MyOne silane beads (Thermo Fisher Scientific, Waltham, MA). The cDNA was amplified, SPRIselect (Beckman Coulter, Brea, CA) cleaned and quantified, and then enzymatically fragmented and size selected using SPRIselect beads to optimize the cDNA amplicon size prior to library construction. An additional round of double-sided SPRI bead cleanup was performed after end repair and A-tailing. Another single-sided cleanup was done after adapter ligation. Indexes were added during PCR amplification and a final double-sided SPRI cleanup was performed. Libraries were quantified by KAPA quantitative PCR for Illumina adapters (Roche, Pleasanton, CA) and size was determined by Agilent TapeStation D1000 tapes. Read 1 primer sequences were added to the molecules during GEM incubation. P5, P7, and sample index and read 2 primer sequences were added during library construction via end repair, A-tailing, adaptor ligation, and PCR. Libraries were generated with unique sample indices for each sample.