title: GSM6745655: 10x multiome (RNA) on the mouse liver using the NST protocol; Mus musculus; RNA-Seq
library_construction_protocol: For the “Multiome-10x_Fresh_Mouse-4” sample 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)). For the “Multiome-NST_Fresh_Mouse-5” sample nuclei isolation we used a modified protocol from the Slyper et al. (2020). 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 (Salt-tris solution - 146 mM NaCl, 10 mM Tris 7.5, 1 mM CaCl2, 21 mM MgCl2, 0.2% IGEPAL CA-63, 0.01% BSA, 0.2 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 1 mL of homogenization buffer and 3 mL salt-tris solution buffer (146 mM NaCl, 10 mM Tris 7.5, 1 mM CaCl2, 21 mM MgCl2). The tissue material was spun down at 500 x g for 5 min. The obtained pellet, after supernatant removal, was resuspended in 1.5 mL salt-tris solution buffer with 0.2 U/µL RNasin Plus RNase Inhibitor (Promega). The tissue material was spun down at 500 x g for 5 min. The obtained pellet, after supernatant removal, was resuspended in 1.5 mL wash buffer (1x PBS, 1% BSA and 0.2U/μL RNasin Plus RNase Inhibitor (Promega)). The wash step was repeated one more time. The final pellet was resuspended in 500 µL wash buffer, filtered, stained with DAPI (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. The nuclei pellet was resuspended in diluted nuclei buffer (1x Nuclei buffer Multiome kit (10x Genomics)), 1 mM DTT, 1 U/µL RNasin Plus RNase Inhibitor (Promega)). 9 μL of sample was mixed with 1 μL of arginine orange/propidium iodide (AO/PI) stain, loaded onto a LUNA-FL slide and visualized with the LUNA-FL Automated cell counter for nuclei yield, morphology and presence of clumps/debris. 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 NovaSeq6000 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.