title: GSM3400452: 7_wo_B6_males_pool1; Mus musculus; RNA-Seq
library_construction_protocol: The pituitary glands of six individually-housed 7-week-old male C57BL/6 mice were dissected, and the tissue dispersed as previously described14-16, as two pools of three glands each.  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 3' library generation was performed on the 10x Genomics Chromium Controller following the manufacturers protocol for the v2 reagent kit (10X Genomics, Pleasanton, CA, USA)10.  Cell suspensions were loaded onto a Chromium Single-Cell A chip along with reverse transcription (RT) master mix and single cell 3' gel beads, aiming for 10,000 cells per channel.  For each pool, 17,400 cells with 71-82 % viability were encapsulated into emulsion droplets at a concentration of 700-1200 cells/ul, which targets 10,000 single cells with an expected multiple cell rate rate of 7.6 %.  Following generation of single-cell gel bead-in-emulsions (GEMs), reverse transcription was performed and the resulting post GEM-RT product was cleaned up using DynaBeads MyOne Silane beads (ThermoFisher Scientific, Waltham, MA, USA).  The cDNA was amplified, SPRIselect (Beckman Coulter, Brea, CA, USA) cleaned and quantified 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 is performed after end repair and A-tailing.  Another single-sided cleanup is done after adapter ligation.  Indexes were added during PCR amplification and a final double-sided SPRI cleanup was performed.  Libraries were quantified by Kapa qPCR for Illumina Adapters (Roche) and size was determined by Agilent tapestation D1000 tapes.  Read 1 primer sequence were added to the molecules during GEM incubation.  P5, P7 and sample index and read 2 primer sequence were added during library construction via end repair, A-tailing, adaptor ligation and PCR.  Libraries were generated with unique sample indices (SI) for each sample. Libraries were sequenced on a HiSeq 4000 (Illumina, San Diego, CA, USA), using a HiSeq 4000 PE Cluster Kit with HiSeq 4000 SBS 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 x 8 x 98 (115) cycles for Read 1, Index and Read 2, respectively.  Cell Ranger Single Cell Software Suite 2.1.1 was used to perform sample demultiplexing and barcode processing at the University of Michigan Biomedical Core Facilities DNA Sequencing Core.
attributes.tag: GEO Accession
attributes.value: GSM3400452