GSM3758118: skin samples from subject o2; Homo sapiens; RNA-Seq
For each experiment, 4-mm punch biopsies were obtained from healthy whole skin specimens, immediately after resection from the inguinoiliac region of five male subjects. Samples were kept in MACS Tissue Storage Solution (Miltenyi Biotec) for no longer than 1 h before their enzymatical and mechanical dissociation with the Whole Skin Dissociation kit for human material (Miltenyi Biotec) and the Gentle MACS dissociator (Miltenyi Biotec), following the manufacturer's instructions. Cell suspensions were then filtered through 70-µm cell strainers (Falcon) and depleted of apoptotic and dead cells with the Dead Cell Removal Kit (Miltenyi Biotec). Sequencing libraries were subsequently prepared following the Drop-seq methodology (Macosko et al., 2015), using a Chromium Single Cell Controller and the v2 chemistry from 10X Genomics. Thus, approximately 20,000 cells per sample were mixed with the retrotranscription reagents and pipetted into a Chip A Single Cell, also containing the Single Cell 3' Gel Bead suspension and Partitioning Oil. The Chip was subsequently loaded into a Chromium Single Cell Controller (10X Genomics) where the cells were captured in nanoscale droplets containing both the reagents needed for reverse transcription and a gel bead. Resulting Gel Bead-In-EMulsions (GEMs) were then transferred to a thermocycler in order to perform the retrotranscription following the manufacturer's protocol. Each gel bead contained a specific 10X Genomics barcode, an Illumina R1 sequence, a Unique Molecular Identifier (UMI) and a poly-dT primer sequence. Therefore, from poly-adenylated mRNA the reaction produced full-length cDNA with a unique barcode per cell and transcript, which allowed tracing back all cDNA coming from each individual cell. Following an amplification step, cDNA was further processed by fragmentation, end repair and A-tailing double-sided size selection using AMPure XP beads. Finally, Illumina adaptors and a sample index were added through PCR using a total number of cycles adjusted to the cDNA concentration. After sample indexing, libraries were again subjected to double-sided size selection. Quantification of the libraries was carried out using the Qubit dsDNA HS Assay Kit (Life Technologies), and cDNA integrity was assessed using D1000 ScreenTapes (Agilent Technologies). Paired-end (26+74bp) sequencing (100 cycles) was finally performed with a HiSeq 4000 device (Illumina).
GEO Accession
GSM3758118