title: GSM7829700: E8.5-scATAC-Library; Mus musculus; ATAC-seq
library_construction_protocol: Freshly harvest mouse embryonic hearts were digested into single cells using 1 mL of 0.25% trypsin-EDTA at 37 degrees Celsius for 5 minutes with gentle trituration using a 1000 uL pipette. Equal volume of collagenase mixture (collagenase A and B) dissolved in HBSS without calcium and 40% FBS was then added to the trypsin mixture for an additional 20 minutes. Cells were triturated using a 1000 uL pipette throughout incubation period and were then pelleted by centrifugation for 5 minutes at 200g at 4 degrees Celsius. Cells were then washed and resuspended in cold PBS + 0.04% BSA. Nuclei were extracted from cell suspensions by following protocol CG000366 Rev D provided by 10X Genomics. Cells were centrifuged at 500 rcf for 5 minutes at 4°C and incubated in a diluted (0.1X) lysis buffer on ice for 5 minutes. They were subsequently washed twice in wash buffer and nuclei were centrifuged again at 500 rcf for 5 minutes. Nuclei pellets were then resuspended in a diluted nuclei buffer and subsequent steps for scRNA-seq and scATAC-seq capture were conducted using the 10X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev F) protocol. Input nuclei concentration was adjusted to achieve an input nuclei number of 10,000 cells per timepoint according to the manufacturer's protocol. Samples were incubated with transposition mix containing ATAC enzyme and buffer for 60 minutes at 37°C as specified by the protocol. GEMs were generated using three separate gel bead tubes, one per sample. Post-GEM incubation cleanup was performed per the manufacturer's protocol. Following pre-amplification PCR, 40 µl of pre-amplified sample was used to generate the ATAC library, while 35 µl sample was used to construct the cDNA and gene expression libraries. Library quality and average fragment size were assessed using a Bioanalyzer (Agilent, G2939A) High Sensitivity DNA chip (Agilent, 5067-4626). Libraries were sequenced with Illumina NovaSeq 6000 at 20,000 read pairs per nuclei for the gene expression library and 25,000 read pairs per nuclei for the ATAC library.