GSM8676660: Papain condition, multiome, ATAC; Mus musculus; ATAC-seq
Single-cell suspensions of hematopoietic and non-hematopoietic lung cells were prepared using distinct digestion protocols. Hematopoietic cells were isolated after a 45-minute digestion at 37°C in digestion buffer (RPMI 1640 supplemented with 1 mg/ml collagenase D, 1 U/ml DNase I, and 25 µg/ml liberase TL). Following digestion, CD45+ hematopoietic cells were enriched via positive magnetic sorting. Non-hematopoietic cells were prepared by digesting lung tissue for 45 minutes at room temperature (RT) in PBS containing 25 mg/ml dispase and 1% low-melting-point agarose. The lungs were subsequently transferred to RPMI 1640 media with 2.4 U/ml DNase I, teased apart, and incubated on a shaker at 37°C for 10 minutes. Non-hematopoietic cells were enriched via negative magnetic sorting for CD45-. Purified cells were pooled together to proceed with scMulti-omics experiments. Library was performed using the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) according to manufacturer's instructions. Briefly, nuclei suspensions were incubated with a transposase, which fragmented the DNA in open regions of the chromatin and added the adapter sequences to the ends of the DNA fragments. The transposed nuclei were loaded onto Chromium Next GEM chip with partitioning oil and barcoded single-cell gel beads, followed by PCR amplification. The ATAC library and gene expression library were then prepared separately. Libraries were then pooled evenly and paired-end sequencing was performed on an Illumina NextSeq 550 Instrument. scMulti-omics