GSM7421540: Prurigo nodularis patient 42 lesional; Homo sapiens; RNA-Seq
Tissues were digested in a pre-warmed dissociation solution (3 mg ml−1 collagenase I (Worthington) and 4 mg ml−1 dispase II (Roche) in PBS) in conical tubes positioned horizontally in a shaking water bath for 1.5 h min at 180 r.p.m. at 37 °C. Samples were then lightly triturated with sterilized Pasteur pipettes (VWR) until the skin samples broke up. They were then run through a 40 μm filter (VWR) and rinsed with 3 ml TSPC medium (αMEM (Gibco) supplemented with 20% FBS, 1%PS) to inactivate the enzymes. Cells were centrifuged at 300g for 30 min at 4 °C. The cell pellet was washed twice with 1 ml PBS and spun for 10 min at 300g at 4 °C.  Cells were then resuspended in HBSS at a concentration of ~1,000 cells/μl.  Cell viability was assessed with Trypan blue exclusion on a Countess II (ThermoFisher Scientific) automated counter and showed a >85% viability. Library was performed according to the manufacter's instructions (Chromium Single Cell 5' Reagent Kits User Guide (v1.1 Chemistry) 10x Genomics). Briefly, a maximum volume of 37.8 uL/sample was used for processing to target up to 10,000 cells.  Cells were combined with RT reagents and loaded onto 10X Next GEM Chip G along with 5' v1.1 gel beads.  The NextGEM protocol was run on the 10X Chromium Controller to create GEMs, composed of a single cell, uniquely barcoded gel bead, and RT reagents.  Approximately 100uL of emulsion is retrieved from the chip and incubated (45 min at 53C , 5 min at 85C, cool to 4C), generating barcoded cDNA from each cell.  The GEMs are broken using Recovery Agent and cDNA is cleaned, following manufacturer's instructions using MyOne SILANE beads.  The cDNA is amplified for 13 cycles (45sec @ 98C, 13 cycle: 20sec @98C, 30sec @67C, 1min @72C; 1min @ 72C, cool to 4C).  Samples are cleaned using 0.6X SPRIselect beads and the quality control analyses (QC) is completed using Qubit and Bioanalyzer to determine size and concentrations.  Next, 10uL of amplified cDNA is carried into library prep.  Fragmentation, end repair, and A-tailing are completed (5 min @ 32C, 30min @ 65C, cool to 4C), and samples are cleaned up using double sided size selection (0.6X, 0.8X) with SPRIselect beads.  Adaptor ligation (15min @ 20C, cool to 4C), 0.8X cleanup and amplification are performed, with PCR using unique i7 index sequences.  Libraries undergo a final cleanup using double sided size selection (0.6X, 0.8X) with SPRIselect beads.    Library QC for the gene expression libraries is performed using Qubit, Bioanalyzer, and KAPA library quantification qPCR kit.  Libraries are sequenced on the Illumina NovaSeq 6000 using v1.5 kits, targeting 50K reads/cell for GEx libraries, at read lengths of 26 (R1), 8 (i7), 91 (R2).  Demultiplexing and FASTQ generation is completed using Illumina's BaseSpace software.