title: GSM4219538: pituitary gland, Hesx1Cre_dnRAR; Mus musculus; RNA-Seq
library_construction_protocol: Pituitary cells were dissociated as previously described in Cheung et al. 2018 Endocrinology 159(12):3910-3924.  The pituitary glands were dissected and the tissue dispersed as previously described.  Briefly, the dissected tissue is placed into an enzyme mix containing 0.5% w/v collagenase type 2 (Lorne Laboratories, UK), 0.1x trypsin (Invitrogen), 50 mg/ml DNase I (Worthington Biochemical, USA), and 2.5 mg/ml Fungizone (Invitrogen) in Hank's balanced salt solution (HBSS) (Invitrogen) for 4 hrs at 37°C.  The tissue and cells are gently triturated, counted on a hemocytometer, pelleted at 200g for 5 min, and resuspended at ~1000 cells per ml in 0.4% bovine serum albumin (BSA)/HBSS. Single-cell libraries were constructed as previously described in Cheung et al. 2018 Endocrinology 159(12):3910-3924.  From that article: Single-cell 3′ library generation was performed on the 10x Genomics Chromium Controller following the manufacturer's protocol for the v2 reagent kit (10x Genomics) (10). Cell suspensions were loaded onto a Chromium Single Cell A Chip along with reverse transcription master mix and single-cell 3′ gel beads, aiming for 10,000 cells per channel. For each pool, 17,400 cells with 71% to 82% viability were encapsulated into emulsion droplets at a concentration of 700 to 1200 cells per μL, which targets 10,000 single cells with an expected multiple cell rate of 7.6%. 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. Libraries were sequenced on a HiSeq 4000 (Illumina, San Diego, CA) using a HiSeq 4000 paired-end cluster kit with HiSeq 4000 sequencing by synthesis kit (100 cycles, reagents, loaded at 200 pM) following Illumina's denaturing and dilution recommendations. PhiX was spiked into the load at 1%. The run configuration was 26 × 8 × 98 (115) cycles for read 1, index, and read 2, respectively.
attributes.tag: GEO Accession
attributes.value: GSM4219538