title: GSM9460820: Sample 19, lesional scalp; Homo sapiens; RNA-Seq
library_construction_protocol: Frozen tissues were rapidly thawed, minced, and subjected to two sequential enzymatic digestions in Liberase TL (2 mg/ml stock diluted in PBS) at 37 °C with gentle rotation to obtain a single-cell suspension, with an optional brief secondary digestion in 0.05% Trypsin for highly cohesive samples. Following digestion, released cells were filtered through FBS-prewetted 40–70 µm strainers into RPMI containing 20% FBS, washed with cold PBS, and pelleted by low-speed centrifugation at 4 °C before final resuspension in RPMI with 2% BSA and RNase inhibitor for 10x Genomics GEM generation. Cell concentration and viability were assessed using a LUNA automated cell counter with Acridine Orange/Propidium Iodide staining prior to loading. Library preparation was performed using the Chromium NextGEM Single Cell 5' Kit v2 (Dual Index) to generate 5′ gene expression libraries from amplified full-length cDNA produced after GEM-RT and cDNA amplification according to manufacturer's protocol. The amplified cDNA was first enzymatically fragmented, followed by combined end repair and A-tailing reactions, with reaction products subjected to a double-sided SPRIselect cleanup to enrich for appropriately sized inserts. Size-selected fragments then underwent adaptor ligation to add truncated Illumina sequencing adaptors, another SPRIselect cleanup to remove excess adaptors and small fragments, and a final sample index PCR that incorporated P5, P7, i5, and i7 sequences to yield dual-indexed, Illumina-ready 5′ gene expression libraries.