title: GSM6138439: Sample_B3; Homo sapiens; RNA-Seq
library_construction_protocol: Bulk RNA-seq: After 24h or 48 h of culture, NMK was treated with 1 ml of Trizol to isolate RNA for further bulk RNAseq experiments. ScRNA-seq: Single-cell suspensions were generated immediately after manual isolation of the human scalp specimens.  Briefly, the scalp tissue for scRNA-seq was washed in phosphate-buffered saline (PBS) and cut into small pieces. After chopping the HF and DP with a scalpel in a Petri dish, samples were transferred to prepared dissociation solution (2 mg/ml Liberase TL in PBS, Sigma Aldrich) and incubated for 60 min at 37ºC for digestion. The residual tissue was transferred into a dissociation solution containing 0.05% Trypsin and incubated for 30 min at 37ºC for a second round of dissociation. Then dissociated samples was filtered out using a 70-μm cell strainer (#352340, Corning). Following centrifugation (300 x g for 5 min), the supernatant was decanted, and the live cells were resuspended in PBS-0.04% bovine serum albumin (Sigma Aldrich). The samples were centrifuged (200 x g for 5 min) and then resuspended in 1ml PBS-0.04% bovine serum albumin. Live cells were counted and confirmed using LUNA-FL dual fluorescence cell counter (Logos Biosysteomics). Bulk RNA-seq: Libraries preparation for 150bp paired-end sequencing was processed using the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero H/M/R (Illumina Technologies., San Diego, CA, USA), following the protocol. The integrity and overall quality of the total RNA was quantified using a Nanodrop 8000 UV- Vis spectrometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and checked with a BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). After cluster amplification of denatured templated, the prepared libraries were sequenced using Illumina Novaseq6000 (Illumina Technologies., San Diego, CA, USA). ScRNA-seq: To generate single-cell gel bead emulsions, suspensions of skin cells were immediately injected into the Chromium Controller (10x Genomics) targeting 10,000 cells, respectively. Following the manufacturer's protocols, the Chromium Single Cell 3' Reagent V2 Chemistry Kit(10x Genomics) was used to construct single cell libraries. Then, prepared libraries were sequenced on NovaSeq 6000 Sequencing System (Illumina, USA)  targeting 50,000 raw reads per cell.