GSM9061702: human keratinocytes, plate 2; Homo sapiens; OTHER
Cells (> 3 x 106) were harvested with trypsin and cross-linked in suspension by incubating for 10 minutes with 4% paraformaldehyde (PFA) in PBS following a quenching step of 5 minutes with 125 mM Glycine in PBS. Removal of PFA and Glycine occurred through washing twice with a wash buffer (0.1x PierceTM Protein-Free Blocking Buffer from Thermo in PBS). Then, cells were blocked in 500μl blocking buffer (0.5x PierceTM Protein-Free Blocking Buffer, 200 μg/ml boiled salmon sperm DNA, 0.1% Triton-X 100, in PBS) at room temperature for 30-60 min. Pre-stainings were performed at room temperature for 1-2 hours and staining with the conjugate mix occurred overnight at 4°C in 500 μl blocking buffer. After each staining, cells were washed 3x in 5 ml wash buffer. Cells were sorted in single wells with the BD FACSAria SORP flow cytometer (BD biosciences) in 96 well PCR plates containing 1 μl release buffer (10 mM DTT in 15mM Tris, pH 8.8) and 7 µl Vapor-lock (Qiagen). Library preparation was performed as described previously (PMID: 30466065). Briefly, 3 PCR steps were performed to amplify the antibody barcodes and to add barcodes specific for the well and the plate of each cell. The barcoding occurred with the same sequences used in (PMID: 30466065). For the first PCR step 15 cycles were run after adding to each well a 4 µl reaction mix containing the Herculase II Fusion DNA Polymerase (Agilent), dNTPs, 5x Herculase buffer and 0.1μM amplification primers. Directly after the first PCR step, 5 extra cycles were run after adding 1 µl mix containing Herculase buffer 0.2 μM forward amplification primer and 0.2 μM reverse well barcoding primer. Then all material was pooled per plate, Vapor-lock was removed and a clean-up was performed with the QIAquick PCR Purification Kit, followed by an exonuclease 1 treatment to degrade remaining primers and another purification. Another 5 cycles were run in PCR 3 with a 20 µl reaction containing the pooled and purified sample and 0.1 μM plate-barcoding primers. After repeating the clean-up procedure, the libraries were checked on agarose gel and with the Bioanalyzer (Agilent) to confirm the size of the DNA fragments (expected size around 185 bp) and sequenced on an Illumina NextSeq500 machine.