GSM3589976: HODay0replicate2; Mus musculus; RNA-Seq
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). 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 2000-6000 cells per channel. In this experiment, 8700 cells were encapsulated into emulsion droplets at a concentration of 700-1200 cells/ul which targets 5000 single cells with an expected multiplet rate of 3.9%. 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 are added to the molecules during GEM incubation. P5, P7 and sample index and read 2 primer sequence are added during library construction via end repair, A-tailing, adaptor ligation and PCR. Libraries were generated with unique sample indices (SI) for each sample.