title: GSM6674374: SHARESeq_ATAC_T128_3; Homo sapiens; ATAC-seq
library_construction_protocol: Frozen human Bone Marrow Mononuclear Cells (BMMCs, Allcells) were thawed in a 37 °C water bath for 1 min and transferred to a 15 mL centrifuge tube. 10 mL of pre-warmed DMEM with 10% FBS was added to cells drop-wisely. The cells were spun at 400g for 3 min at room temperature. After removing supernatant, the cells were washed twice in 0.5 mL PBS with 0.04% BSA. To deplete neutrophils, the cells were resuspended in 100 μl chilled DPBS with 0.2% BSA and 10 μl of human TrueStain FcX (BioLegend, 422302) and incubated on ice for 10 min to reduce non-specific labeling. The cells were then incubated on ice for another 30 min after adding 0.5 μl of biotin anti-human CD15 antibody (BioLegend, 301913). After immunostaining, 25 μl of MyOne T1 beads were added to the sample to capture the neutrophils for 5 min at room temperature. We then added 900 μl of DPBS with 0.2% BSA to dilute the sample. The sample was placed on a magnet for 3 min and 1 ml of the sample was transferred to a new tube while the sample was on the magnet. The cells were ready for fixation and SHARE-seq experiment. Cells were centrifuged at 300g for 5 minutes and resuspended to 1 million cells/ml in PBSI. Cells were fixed by adding formaldehyde (28906, ThermoFisher) to a final concentration of 1% and incubated at room temperature for 5 minutes. The fixation was stopped by adding 56.1 μl of 2.5M glycine, 50 μl of 1M Tris-HCl pH 8.0, and 13.3 μl of 7.5% BSA on ice. The sample was incubated at room temperature for 5 minutes and then centrifuged at 500g for 5 minutes to remove supernatant. All centrifugations were performed on a swing bucket centrifuge. The cell pellet was washed twice with 1ml of PBSI, and centrifuged at 500g for 5 minutes between washings. The cells were resuspended in PBS with 0.1U/μl Enzymatics RNase Inhibitor and aliquoted for transposition. Following fixation SHARE-seq was performed as previously described in the original SHARE-Seq paper, with the following modifications. To improve transposition, transposition was performed using pre-assembled Tn5 (seqWell, Tagify(TM) SHARE-Seq Reagent). To improve RNA capture, we added polyA to transcripts prior to reverse transcription. To do this, transposed cells (60 μl) were mixed 240 μl of poly(A) mix (final concentration of 1× Maxima RT buffer, 0.25 U/μ