title: GSM8517991: LA450, KT;Stag2 flox, 16 weeks; Mus musculus; Hi-C
library_name: GSM8517991
library_construction_protocol: Tumors were dissociated using collagenase IV, dispase, and trypsin at 37 degrees C for 30 minutes as previously described (40). Cells were stained with DAPI and antibodies against CD45 (30-F11), CD31 (390), F4/80 (BM8), and Ter119 (all from BioLegend) to exclude hematopoietic and endothelial cells. FACSAria sorters (BD Biosciences) were used for cell sorting. In situ Hi-C libraries were prepared from FACS-sorted neoplastic cells with low input ranging from 2 x 105 to 4 x 105 cells. In detail, cells were first pelleted at 300 G and resuspended in 1 mL Room Temp PBS containing 3% BSA, then fixed as previously described, (Rao et al. Cell 2014) only with a higher centrifuge speed at 2,500 G for pelleting the fixed cells. Fixed cell pellets were processed through the standard Arima Hi-C kit (Catalog # A510008), until the step of Covaris DNA sonication to an average size of 400 bp. Sheared DNA was purified and size-selected by a two-step AMPure XP bead (Beckman Coulter #A63882) cleanup at 0.6x and 1.0x, and then inputted into the 2S Plus DNA Library Kit from Integrated DNA Technologies (Catalog #10009878) for low-input library preparation, until the step before library PCR amplification. Libraries underwent a biotin pulldown using streptavidin to enrich chromatin interactions before an initial five-cycle amplification was performed and a qPCR quantification determining the number of additional cycles required for each library to reach a final concentration around 20 nM in 20 µL. Amplified libraries were then examined by Agilent 4200 TapeStation for confirmation of size distribution before submission for sequencing. Libraries were first sequenced using the Illumina MiniSeq PE50 for quality control (analysis described in the next section), and then deeply sequenced using the Illumina NovaSeq PE50 aiming at ~4  x 108  read pairs for each library that passed the MiniSeq QC.
identifiers.namespace: GEO
identifiers.id: GSM8517991_r1