GSM9109632: Plate2_TF_TCF3_P2E8_dose0.75_rep1; Homo sapiens; ATAC-seq
OmniATAC: follows standard OmniATAC protocol in Corces et al Nat Methods 2017. RoboATAC: A 96-well plate of cells in media was directly placed on an Agilent Bravo liquid handling platform to begin the automated RoboATAC workflow. Briefly, after removing the media, each well of roughly 50,000 HEK293T cells were lifted in 30uL trypsin for 3 min at 37C, neutralized with 120uL media, and pulled down from trypsin:media solution by binding to 10uL Concanavalin A beads for 10min at room temperature and magnetically separating and removing the supernatant. We added 50uL cold OmniATAC lysis buffer directly to the nuclei pellet, incubated for 3min at 4C, then quenched with 150uL cold OmniATAC wash buffer, magnetically separated and removed the supernatant, added 50uL OmniATAC transposition buffer directly to the pellet, and incubated at 37C for 30min with 1300rpm shaking. For suspension cells, we skipped the trypsinization steps and proceeded directly to ConA bead binding to cells in media. OmniATAC: follows standard OmniATAC protocol in Corces et al Nat Methods 2017. RoboATAC: To clean up the transposition product, we added 165uL Qiagen buffer RLT directly into the transposition mix, magnetically separated the supernatant from the ConA beads, and transferred the supernatant containing the transposition product into a new plate. We added 10uL Silane beads and 225uL 100% Isopropanol into each well of transposition product, incubated for 2min at room temperature, magnetically separated and removed the supernatant. We washed the pellet twice with 300uL 70% Isopropanol and air dried the pellet for 5 min at room temperature. We eluted the cleaned transposition product with 20uL of H2O for 5min at room temperature, then performed PCR following standard OmniATAC setup for 10 cycles total (72C x 5m, 98C x 30s, then 10 cycles of 98C x 10s + 63C x 30 + 72C x 30s, hold at 4C). The PCR products were cleaned using SPRI beads at a 1.2x ratio following standard SPRI cleanup protocol. TF dose RoboATAC libraries were converted for sequencing on the Ultima Genomics UG 100 sequencer via a conversion PCR reaction (Ultima Protocol Guide D1001055 Rev 01) using 10ng of input DNA, sample-specific UG indexing primers targeting the Nextera Read 1 sequence, and a UG universal primer targeting the Nextera Read 2 sequence. Converted libraries were purified using a SPRI bead ratio of 1.5x.