GSM4973304: LCH97_Skin; Homo sapiens; RNA-Seq
LCH skin samples were obtained with informed consent from patients undergoing diagnostic biopsies. Control epidermal LCs were isolated from discarded skin after the elective circumcisions from patients<18year.   Child skin samples were first incubated in RPMI 1640 medium with 1.2 U/ml dispase II (Roche, Indianapolis, IN) at 4 °C for 8 h prior to separation of the epidermal layer. After separation, the epidermal layer was cut into pieces and digested with 0.25% trypsin-EDTA (Gibco) containing 0.25 mg/ml DNase I (Sigma DN25) for 30 min at 37 °C in a humidified incubator. After neutralization, the epidermal layer was passed through a 70-μm cell strainer by grinding, and then washed with PBS. The following antibodies were used to label the cells: CD45 (BV421, 563879 BD, lot 9066960), 7-amino-actinomycin D (7-AAD) (PerCP-Cy5.5, 00699350 eBioscience, lot 1910559), CD207 antibodies (PE, 564727 BD, lot 8135683) and CD1a antibodies (APC, 559775 Biolegend, lot 8164562).   Blood samples were treated with lysis buffer (BD) to remove erythrocytes, and then were subjected to staining using: CD45 (BV421, 563879 BD, lot 9066960), 7-amino-actinomycin D (7-AAD) (PerCP-Cy5.5, 00699350 eBioscience, lot 1910559), HLA-DR (PE, 307606 Biolegend, lot B289372), CD14 (BV786, 563698 BD, lot 8351911), CD3 (APC-Cy7, 300426 Biolegend, lot B289558), CD19 (APC-Cy7, 557791 BD, lot 9064665), CD56 (APC-Cy7, 362512 Biolegend, lot B281874), CD303/BDCA2 (PE-Cy7, 354214 Biolegend, lot B268476) and CD11c (FITC, 561355 BD, lot 8054660).  After staining for 30min in 4°C, cells were washed and resuspended in PBS, and then  were sorted using an Aria 2 Flow Cytometer (BD Bioscience). Data were analysed using BD FACSDIVA V8.0.1 and Flowjo (V10). Single-cell capturing and downstream libraries were constructed following a modified single-cell tagged reverse transcription (STRT) protocol as previously reported. Cells in good condition were picked by mouth pipette and transferred to lysis buffer. The reverse transcription reaction was performed using a sample-specific 25-nucleotide (nt) oligo dT primer containing an 8-nt barcode (TCAGACGTGTGCTCTTCCGATCT -XXXXXXXX-DDDDDDDD-T25, where X represents the sample-specific barcode, and D stands for the unique molecular identifier (UMI)). After reverse transcription and second-strand cDNA synthesis, cDNAs were amplified by 16-18 cycles of PCR. Then the barcoded DNAs were pooled together and purified with Agencount AMPure XP beads. Biotinylated pre-indexed primers were used to further amplify the PCR products by another four cycles of PCR to introduce biotin tags into the 3′ ends of the amplified cDNAs. About 300 ng cDNA were ultrasonically processed into 300-bp fragments using Covaris S2 system and enriched with Dynabeads MyOneTM Streptavidin C1 beads. Libraries were constructed using a Kapa Hyper Prep Kit (Kapa Biosystems) and 150-bp paired-end sequencing was performed on the Illumina Hiseq X Ten platform (Novogene).