GSM8140658: Donor 1, ICOS-Low, 24 hour Restimulation; Homo sapiens; OTHER
Sorted samples were pelleted and resuspended in 400uL ChIP Lysis Buffer (1% SDS, 50mM Tris, pH 8, 10mM EDTA) per 5e6 cells. Each 400uL reaction received 16µl NaCl (5M) and was incubated at 66°C overnight. Subsequently, each reaction received 8µl RNAse A (Fisher Scientific #EN0531) and was incubated at 37°C 1h. Then, 8µl Proteinase K (Fisher Scientific #25530049) was added and the samples incubated at 55°C 1h. One phase lock tube (Quantabio, #2302820) per sample was spun at 20,000g 1 minute and received 400µl Phenol:Chloroform:Isoamyl Alcohol (25:24:1). 400µl sample was added to each phase lock tube, shaken vigorously, and centrifuged at 20,000g 25°C 5 minutes. Aqueous phases were transferred to low-binding tubes (Eppendorf, #022431021) and received 40µl of Sodium Acetate (Fisher Scientific #46-033-CI), 1µl GlycoBlue (Invitrogen, #AM9515), and 600µl isopropanol. Samples were vortexed and frozen at -80°C ≥30 minutes. Frozen samples were centrifuged 20,000g 4°C 30 minutes, pellets washed with fresh 70% ethanol, and allowed to air dry for 15 minutes. Genomic DNA pellets were resuspended in Zymo DNA elution buffer (Zymo, #D3004-4-10) and reconstituted at 65°C for 1 hour, or until dissolution. Sequencing libraries were generated using 3.75ug genomic DNA per 50uL PCR reaction with 0.25uM CM_oligo_4 and 0.25uM unique p7 reverse primer as in CM_oligo_5 (see Supplementary Information). PCR reactions were run with the following parameters: 95°C 1', [95°C 30”, 60°C 30”, 72°C 30”] x 28, 72°C 10', 4°C hold. Amplicons were purified with DNA Clean & Concentrator-25 kits (Zymo Research #D4033)100. One sample (Donor 2 Tconv cells, ICOS screen) was re-indexed before sequencing. Pooled libraries were sequenced with a custom sequencing primer CM_oligo_6 on an Illumina NextSeq500 instrument.