GSM8640240: D1 RNA 2.5h; Homo sapiens; RNA-Seq
Nuclei were prepared according to the modified 10XGenomics  protocol: 100ul of cold lysis buffer (10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2, 1% BSA, 0.025% Tween-20,  0.025% Nonidet P40,  0.00125% Digitonin, 1mM DTT, 1 U/µl RNase inhibitor (Roche)) were added to cell pellets. Samples were incubated for 40s on ice, washed with 1ml of cold wash buffer (10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2, 1% BSA, 0.025% Tween-20, 1mM DTT, 1 U/µl RNase inhibitor (Roche)) and filtered with Flowmi cell strainer (Bel-Art H13680-0040). The Chromium Next GEM Single Cell Multiome ATAC + Gene Expression assay was performed according to the manufacturer's instructions (10x Genomics) with the kit Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle (PN-1000283). Briefly, nuclei suspended in the multiome buffer were combined with a transposition mix and incubated for one hour. The transposition reaction was combined with the kit's master mix and loaded together with partitioning oil and gel beads into the Chromium Next GEM Chip J Single Cell Kit (PN-1000234) to generate a gel bead-in-emulsion (GEM). The GEMs were then incubated producing 10x barcoded DNA from the transposed fragments (for ATAC) and 10x barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). Next the GEMs were broken, the barcoded DNA and cDNA were purified, and the purified products were subjected to a pre-amplification PCR step . The pre-amplified material was then used as starting material for ATAC and RNA library construction per 10x protocol. ATAC libraries and GEX libraries were pooled separately. and sequenced at CCHMC DNA core.