GSM7520491: 10x multiome (ATAC) on the mouse liver (rep 1); Mus musculus; OTHER
We used a modified protocol from the Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing Protocol (CG000375) from 10x Genomics. Briefly, 100 mg of fresh mouse liver tissue was chopped and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer (10 mM NaCl, 10 mM Tris 7.4, 3 mM MgCl2, 0,1% IGEPAL CA-63, 1 mM DTT, 1 U/µL of Protector RNase inhibitor (Sigma)). The tissue was homogenized with 5 strokes of pestle A and 10 strokes of pestle B until a homogeneous nuclei suspension was achieved. The resulting homogenate was filtered through a 70-μm cell strainer (Corning). The homogenizer and the filter were rinsed with an additional 0.5 μL of homogenization buffer. The tissue material was spun down at 500 x g for 5 min and the supernatant was discarded. The tissue pellet was resuspended in wash buffer (1% BSA in PBS + 1 U/µlL of Protector RNase inhibitor (Sigma)). Nuclei were stained with 7AAD (Thermo Fisher Scientific) and viability sorted on a BD FACS Fusion into 5 mL low bind Eppendorf tube containing BSA with RNase inhibitor. The sorted nuclei were spun down at 500 x g for 5 min and the supernatant was discarded. Next, the nuclei were permeabilized by resuspending the pellet in 0.1x lysis buffer (10 mM NaCl, 10 mM Tris 7.4, 3 mM MgCl2, 0.1% IGEPAL CA-63, 0.01% Digitonin, 1% BSA, 1 mM DTT, 1 U/µL of Protector RNase inhibitor (Sigma)) and incubated on ice for 2 min. 1 mL Wash Buffer (10 mM NaCl, 10 mM Tris 7.4, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 1 U/µL of Protector RNase inhibitor (Sigma)) was added. The nuclei were spun down at 500 x g for 5 min and the supernatant was discarded. The nuclei pellet was resuspended in diluted nuclei buffer (1x Nuclei buffer Multiome kit (10x Genomics)), 1 mM DTT, 1 U/µL of Protector RNase inhibitor (Sigma)). Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and NextGEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) according to the manufacturer's protocol. In brief, the single mouse liver nuclei were incubated for 60 min at 37°C with a transposase that fragments the DNA in open regions of the chromatin and adds adapter sequences to the ends of the DNA fragments. After generation of nanolitre-scale gel bead-in-emulsions (GEMs), GEMs were incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 37°C for 45 min, 25°C for 30 min and hold at 4°C. Incubation of the GEMs produced 10x barcoded DNA from the transposed DNA (for ATAC) and 10x barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). Next quenching reagent (Multiome 10x kit) was used to stop the reaction. After quenching, single-cell droplets were dissolved, and the transposed DNA and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To fill gaps and generate sufficient mass for library construction, the transposed DNA and cDNA were amplified via PCR: 72°C for 5 min; 98°C for 3 min; 7 cycles of 98°C for 20 s, 63°C for 30 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. The pre-amplified product was used as input for both ATAC library construction and cDNA amplification for gene expression library construction. Illumina P7 sequence and a sample index were added to the single-strand DNA during ATAC library construction via PCR: 98°C for 45 s; 7-9 cycles of 98°C for 20 s, 67°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 4°C. The sequencing-ready ATAC-seq library was cleaned up with SPRIselect beads (Beckman Coulter). Barcoded, full-length pre-amplified cDNA was further amplified via PCR: 98°C for 3 min; 6-9 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 5-16 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready GEX library was cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip. All 10x Multiome ATAC libraries were sequenced on NextSeq2000 instruments (Illumina) with the following sequencing parameters: 50 bp read 1 – 8 bp index 1 (i7) – 16 bp index 2 (i5) - 49 bp read 2. All 10x Multiome GEX libraries were sequenced on NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2.