GSM6190914
At time of analysis, mice were euthanized, back skin was dissected from mice, excess scapular and inguinal fat was removed, and wounds plus adjacent tissue was excised using an 8 mm biopsy punch. An 8 mm biopsy punch was used to collect unwounded back skin as a day 0 or unwounded control. Four to six 8 mm biopsy punches were pooled from unwounded back skin to collect sufficient cells for downstream analysis. On ice, tissue was finely minced with scissors and then placed in a 2 ml tube containing 1 ml of digestion medium (2 mg/ml collagenase XI, 0.5 mg/ml hyaluronidase, 0.1 mg/ml DNase in RPMI with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM beta-mercaptoethanol). The tube was placed horizontally in a bacterial shaker for 45 min at 37ºC and 225 rpm. The sample was then filtered through a 100 µm filter and washed with 10 ml cold RPMI. The generated single cell suspension was then processed for further depending on analysis method. The pool was then split over 5 lanes of a 10X 3' NEXTGEM chip before encapsulation with a targeted cell number of 12,000 per lane. Following the MULTI-Seq library construction protocol, additive primer was spiked in during the cDNA amplification step as described in McGinnis et al5, the supernatant was reserved following SPRI cleanup and separately amplified. Finally, libraries were pooled for sequencing using a 1:10 molar ratio of LMO : gene expression (GEX) libraries with approximately equal representation from each lane. Pooled samples were then sequenced using 1 lane of a S4 flowcell for a target of ~3B reads. GEX libraries had total read counts of (493M, 515M, 495M, 452M, 403M) for lanes (1,2,3,4,5) respectively, showing similar order of magnitude representation in read counts. The estimated sequencing saturation ranged from 58-64% (calculated via CellRanger), indicating sequencing depth did not vary appreciably between lanes. LMO libraries had total read counts of (37M, 39M, 38M, 36M, 32M) also showing stable representation from all 5 lanes.