GSM5464270: Acute_2; Homo sapiens; ATAC-seq
50,000 monocytes from HC, acute and post-COVID-19 patients were isolated and cryopreserved in 10% DMSO + 90% SF (n = 3 for each condition). After rapidly unfrozen, cells were counted, lysed (10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.1% TritonX-100 for 5 min at 4ºC and centrifuged at 800 x g. Supernatant was discarded and nuclei were resuspended in transposase reaction buffer with transposase5 (1 µl Tn5 / 10.000 cells) and incubated for 30 min at 37 ºC. Later, SDS at a final concentration of 0.1% was added to stop the reaction (30 min, 40°C) and DNA was isolated with KAPA Pure beads (SPRI beads). Briefly, lysates were incubated with SPRI beads (2x), placed on the magnet, washed twice with 70% ethanol, and air-dried. DNA was eluted with elution buffer (EB) and its concentration measured with Qubit kit (Invitrogen). For DNA library generation, qPCR with i5 and i7 primers (NEXTERA barcoding) was performed (98ºC 2´, 98ºC 20¨, 63ºC 30¨, 72 ºC 1´, 4ºC ∞; 14 cycles). Then, a size cut off was conducted. First, amplified DNA was incubated with 0.5x concentration of SPRI beads. Then, beads were discarded and supernatant was incubated with 1.8x concentration of SPRI beads. Beads were washed twice with 70% ethanol, eluted with EB, and measured with Qubit. Finally, Agilent HS Bionalyzer was performed to assess peak sizes and percentages. Libraries were applied to an Illumina flow cell for cluster generation (PE Cluster Kit cBot) and sequence-by-synthesis paired reads of 50 and 91 base length using the SBS Kit (Illumina) were generated on the HiSeq 4000 following the standard sequencing protocol. Reads were further processed using the bcl2fastq package (Illumina) to split reads according to adapter indexes and produce fastq files. ATAC-Seq was performed at the Genomics Unit of the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid.