title: GSM7596768: FoxP3_Mutant_scATAC; Mus musculus; ATAC-seq
library_construction_protocol: Treg were sorted from mice of the appropriate genotype and desired organ, as detailed in experimental design, into DMEM + 5% FCS in DNA Lo-Bind tubes for input in to scATAC-seq/ASAP-seq as detailed in the next step. For experiments with multiple conditions per scATAC run, we hashtagged cells using a modification of the ASAP-seq strategy (Mimitou et al, Nature Biotech, 2021) for low cell input primary cell samples. Before sorting, cells were hashtagged with TotalSeqA DNA-barcoded hashtag antibodies at the same time as staining with fluorophore-conjugated antibodies (BioLegend). Cells were sorted into DMEM + 5% FCS in DNA Lo-Bind tubes (Eppendorf, cat # 022431021). After spinning down for 5 min at 500g in a refrigerated centrifuge at 4C, cells were resuspended in 100 μl chilled 0.1x Omni Lysis buffer (1x Omni Lysis buffer (10mM Tris-HCl, 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP40 substitute/IGEPAL, 0.01% Digitonin, 1% BSA in nuclease free water) diluted 1:10 in Wash/Lysis Dilution Buffer (10mM Tris-HCl, 10mM NaCl, 3 mM MgCl2, 1% BSA in nuclease free water)), gently mixed by pipetting and incubated on ice for 6.5 min. Following lysis, 100 μl chilled wash buffer was added and gently mixed by pipetting. Cells were spun down for 5min at 500g at 4C, all but 5 μl of supernatant was removed, and 45 μl of chilled 1x nuclei buffer (10x Genomics) was added without mixing. After one more centrifugation step at 500g, 4C for 5 min, supernatant was removed, and samples were resuspended in 7ul 1x nuclei buffer for cell counting and input into transposition, barcoding, and library preparation according to the Chromium Next GEM Single Cell ATAC manual (10x Genomics).   Modifications to the original 10X protocol were made as described in the original ASAP-seq publication and as detailed at https://citeseq.files.wordpress.com/2020/09/asap_protocol_20200908.pdf. Briefly, 0.5 μl of 1uM BOA bridge oligo was spiked into the barcoding reaction. During GEM incubation, an additional 5 min incubation at 40C was added to the beginning of the protocol. 43.5 instead of 40.5 μl of Elution Solution I was added during silane bead elution to recover 43 μl. 40 μl was used for SPRI clean up as indicated in the protocol, while 3 μl was set aside. During SPRI cleanup, the supernatant was saved. The bead bound fraction was processed as in the protocol, while for the supernatant fraction, 32 μl SPRI was added for 5 min. Beads were collected on a magnet, washed twice with 80% ethanol, and eluted in 42 μl EB. This 42 μl was combined with the 3 μl set aside from the previous step as input into the HTO indexing reaction. HTO Indexing PCR was run with partial P5 and indexed Rpxx primers as: 95C 3 min, 12-14 cycles of (95C 20 sec, 60C 30 sec, 72C 20 sec), 72C 5 min. The PCR product was cleaned up with 1.6X SPRI purification for quantification and sequencing alongside ATAC libraries.