title: GSM5420672: human fetal skin-12w; Homo sapiens; RNA-Seq
library_construction_protocol: Human fetal skin was dissected and washed with phosphate-buffered saline (PBS; HyClone, Logan, UT, USA: SH30256.01) on ice after removing fat tissue. The skin was cut into small pieces in digestion medium composed of 1 mg/ml collagenase IV (Shanghai Yeasen Biotechnology Co, Shanghai, China; 40510ES60) in RPMI1640 medium (HyClone; C11875500BT), and incubated at 37°C for 40-60 min. The suspension was filtered through a 70-μm cell strainer (BD Biosciences; Franklin Lakes, NJ, USA; 352350) with gently grinding to release cells. After neutralizing the enzymes with FACS buffer (1 PBS with 2% fetal bovine serum), cells were pelleted at 2000 rpm for 7 min at 4°C and resuspended in FACS buffer, and labeled with specific antibodies in FACS buffer for 30 min on ice. After washing, the cells were resuspended in 1 PBS and were confirmed to be living cells by 4', 6-diamidino-2-phenylindole (DAPI) staining for 5 min. CD45+ cells were sorted on a FACSAria III flow cytometer (BD Biosciences), and data analysis was performed using FlowJo v10 software (TreeStar, Ashland, OR, USA). Fetal skin was processed as described above and cell suspensions were labeled with appropriate antibodies against cell surface markers on ice for 30 min, followed by staining for 5 min with DAPI. Cells were obtained on a Celesta cytometer (BD Biosciences) and analyzed using FlowJo v10 (TreeStar). Single cells were obtained as gel bead-in-emulsions (GEMs) using the 10× Genomics (Pleasanton, CA, USA) Chromium platform according to the manufacturer's protocol. Briefly, skin cell suspensions were combined with reverse transcription master mix and loaded onto the 10× Genomics Single Cell 3' chip, and partitioned into GEMs with gel beads precoated with oligonucleotides containing a poly-dT primer sequence to capture released mRNAs, a 16-bp 10× barcode for cell indexing, and a 10-bp unique molecular identifier (UMI) to distinguish between transcripts. After reverse transcription, barcoded cDNAs for each sample were amplified to generate a sufficient volume for library construction using the Single Cell 3' Reagent Kit (v2 or v3 chemistry). Libraries were subjected to 150-bp paired-end sequencing, which was performed by Novogene (Beijing, China) on a NovaSeq 6000 system (Illumina, San Diego, CA, USA).