GSM9693876: Vegfd-OE, Injured, LST; Mus musculus; RNA-Seq
Cells from the excised tissue were isolated based on previously described methods. Briefly, tissue was digested for 45 min in 0.3% Type 1 Collagenase and 0.4% Dispase II (Gibco) in Roswell Park Memorial Institute (RPMI) medium at 37 °C under constant agitation at 120 rpm. Digestions were quenched with 10% FBS RPMI and filtered through 40μm sterile strainers. Cell viability was assessed with Trypan blue exclusion on a Countess II (Thermo Fisher Scientific) automated counter, and samples with >85% viability were processed for further sequencing. 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. Following the generation of single-cell gel bead-in-emulsions (GEMs), reverse transcription was performed, and the resulting Post GEM-RT product was cleaned using DynaBeads MyOne Silane beads (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was amplified, SPRIselect (Beckman Coulter, Brea, CA, USA) cleaned, 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 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 (PN PE-410-1001) with HiSeq 4000 SBS Kit (100 cycles, PN FC- 410-1002) reagents, loaded at 200 pM following Illumina's denaturing and dilution recommendations. The run configuration was 26 × 8 × 98 cycles for Read 1, Index and Read 2, respectively. Library construction was performed according to the manufacturer's instructions for scRNA (Single Cell 3' v3 protocol, 10X Genomics).
GEO
GSM9693876_r1