GSM8049701: Psoriasis_PN_495; Homo sapiens; RNA-Seq
Four-mm full-depth punch biopsies were obtained from lesional and non-lesional skin of five untreated psoriasis patients and three sex- and age-matched healthy donors. Skin biopsies were enzymatically digested with dispase (5 U/ML, Gibco) overnight to separate the epidermis from the dermis, followed by dissociation of the epidermis to single cells using a protocol optimized to achieve partial dissociation (0.5 % trypsin, 1 U ul−1 DNase, Gibco) for 7 min at 37 °C to obtain both single cells and multiplets. In the single-cell suspensions, cells were counted and diluted to 1 × 106 cells per 100 ul with ice-cold FACS buffer, followed by incubation with dye-conjugated antibodies, including anti-CD45 (APC, eBioscience). After sorting into 384-well plates with hypotonic lysis buffer using a SONY SH800S sorter, both CD45-positive and CD45-negative cells were sorted, excluding dead/dying cells, and stored at -80 °C. cDNA synthesis was based on the Smart-seq2 protocol as implemented in (Andrews et al. Nat. Meth. 2021). In summary, single-cell lysates were thawed on ice in 384-well plates, followed by primer annealing at 72°C for 3 minutes. Subsequently, a reverse transcription (RT) mix of 3 ul was added (0.05 ul of Maxima H Minus Reverse Transcriptase [Thermo Scientific, EP0751], 0.125 ul of Recombinant RNase Inhibitor [Takara Bio, 2313B], 1 ul of 5x First strand buffer, 0.25 ul of 100mM DTT [Thermo Fisher 707265ML], 1 ul of 5M Betaine [Sigma-Aldrich, B0300-5VL], 0.03 ul of 1M MgCl2, 0.05 ul of 100uM TSO [IDT 5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′], and 0.495 ul of H2O). The RT reaction was 42°C for 90 minutes, followed by 70°C for 5 minutes and a 4°C hold. Next, 7.5 ul of cDNA pre-amplification mix was added, consisting of 1.068 ul of H2O, 6.25 ul of 2X Kapa HiFi HotStart ReadyMix (Roche 07958927001), 0.125 ul of 10 uM IS_PCR primer (IDT 5′-AAGCAGTGGTATCAACGCAGAGT-3′), and 0.056 ul of Lambda Exonuclease (ThermoFisher EN0561). The PCR program was 37°C for 30 minutes, 95°C for 3 minutes, followed by [98°C for 20 seconds, 67°C for 15 seconds, 72°C for 4 minutes] repeated for 21 cycles, and a final step of 72°C for 5 minutes, with a 4°C hold. Post-PCR, cDNA clean-up was performed using 0.7X volume SPRI beads and a magnetic 384-well rack. For tagmentation, 1 ul of diluted cDNA (150 pg/ul) was mixed with 1.5 ul of tagmentation buffer (0.5 ul TAP-PEG buffer [50mM TAPS, 25mM MgCl2, 40% PEG 8k) with 0.75 ul H2O)] and 0.25 ul of Tn5 (Vasilios 2020), followed by incubation at 55°C for 10 minutes. Upon completion, Tn5 was inactivated by adding 1 ul of 0.1% SDS (Teknova S0287). Barcoding PCR was performed on the same plate by adding 19.5 ul of PCR mix (Roche7958846001, 13.25 ul of H2O, 5 ul of 5X buffer, 0.75 ul of dNTPs, and 0.5 ul of KAPA HiFi DNA Polymerase) and 2 ul of primers. The total volume of 25 ul underwent the following PCR cycling: 72°C for 3 minutes, 95°C for 30 seconds, [95°C for 15 seconds, 55°C for 30 seconds, 72°C for 45 seconds] repeated for 12 cycles, and a 10°C hold. Finally, 2 ul per well was pooled and subjected to SPRI bead clean-up at 0.9X volume twice. The finished libraries were sequenced on an Illumina NextSeq 550 sequence paired-end 37bp with 8bp dual index.