GSM8684426: H23 cells, CHD1, IgG ChIP-seq; Homo sapiens; ChIP-Seq
To isolate primary LUAD cells, KP mice were euthanized and perfused with PBS through the left ventricle of the heart. Micro-dissected tumor tissues using a sterile scalpel were further dissociated by a mixture of dispase II (0.6 U/mL; D4693, Sigma Aldrich), collagenase type IV (0.166 U/mL; 17104019, Thermo Fisher Scientific), and DNase I (10 U/mL; 07900, STEMCELL Technologies) in Dulbecco's Modified Eagle's Medium (DMEM) containing gentamicin (2 μg/mL; 15710064, Gibco) at 37°C for 30-60 min. The cell suspension was filtered through 70 mm EASYstrainer (Greiner Bio-one) and spun at 300 g for 5 min at 4°C. The supernatant was aspirated, and red blood cells were lysed using RBC Lysis Buffer (eBioscience). Cells were then washed with PBS and spun at 300 g for 5 min at 4°C, re-suspended in ice-cold Fluorescence-Activated Cell Sorting (FACS) buffer [2% fetal bovine serum (FBS) in phosphate-buffered saline solution (PBS)], and strained through a 35 mm filter. Sorted GFP+ LUAD cells were seeded in a mixture of 50% Matrigel (Corning, Cat #: CB-40230C) and 50% LUAD media (Advanced DMEM/F12; 12634-010, Gibco), 2% of FBS, 2mM GlutaMAX (35050061, Gibco), 10 mM HEPES, 2 μg/mL of gentamicin, and 100 units/mL of penicillin/streptomycin). Single-cell RNA-seq was performed on 10x Genomics Chromium platform. The scRNA-seq libraries were prepared based on the manufacturer's protocol for 3' end reading. The individual transcriptomes of encapsulated cells were barcoded during RT step and resulting cDNA purified with DynaBeads followed by amplification per manual guidelines. Next, PCR-amplified product was fragmented, A-tailed, purified with 1.2X SPRI beads, ligated to the sequencing adapters and indexed by PCR. The indexed DNA libraries were double-size purified (0.6–0.8X) with SPRI beads and sequenced on Illumina NovaSeq S4 platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher). The raw sequencing data were then demultiplexed and aligned to the mouse genome using 10X CellRanger pipeline. This generated an output matrix of mRNA counts per cell for every gene. We used the filtered matrix for all downstream analysis. For ChIP-seq, For library construction and sequencing, DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. The KAPA HTP Library Preparation Kit (Kapa Biosystems, Cat #: KK8234) was used to prepare Illumina sequencing libraries with 0.02-5 ng of input DNA and 8-14 cycles of PCR.