GSM5034093: KO_1; Mus musculus; RNA-Seq
Enzymatic digestion with collagenase/dispase followed by RBC lysis in ACK buffer and multiple washes in FACS buffer.  Flow cytometric sorting for GFP+/DAPI- populations and post-sort for viability. Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 with NovaSeq: Cellular suspensions were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-Emulsions (GEMs). Single-cell RNA-Seq libraries were prepared using Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 (10x Genomics). The beads were dissolved and cells were lysed per manufacturer's recommendations. GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken and the pooled post-GEM-RT reaction mixtures were recovered and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, PN37002D, ThermoFisher Scientific). The entire purified post GEM-RT product was amplified by PCR. This amplification reaction generated sufficient material to construct a 3' cDNA library.  Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by End Repair, A-tailing, Adaptor Ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification. Sequence data was generated using Illumina's NovaSeq 6000.
GEO Accession
GSM5034093