GSM8884307: Multiome, HFD, 3 weeks, scATAC-seq, Rep1; Mus musculus; ATAC-seq
Mice were sacrificed using cervical dislocation. The pancreas was injected with 3 mL dissociation buffer (0.7 mg/mL Collagenase P (Merck; Lot#33768602), 1X HBSS (ThermoFisher; 14065049), and 0.3 g/L NaHCO3 (Sigma-Aldrich; S5761)) through the common bile duct. The tissue was dissociated in a tube with 2 mL dissociation buffer for 15 minutes at 37°C, then shaken for 1 minute. Dissociation was stopped by adding cold quenching buffer (1X HBSS, 0.3 g/L NaHCO3, and 10% heat-inactivated fetal bovine serum (VIA) (Biochrom; B15-005)) to a total volume of 20 mL.,The tissue solution was centrifuged for 2 minutes at 150 x g at room temperature (RT), and the supernatant was aspirated until 10 mL remained. This wash step was repeated twice. The tissue was further dispersed by pipetting through a 14G venflon 5 times (without the needle). The solution was then centrifuged for 7 minutes at 200 x g at RT, the supernatant removed, and the tissue pellet resuspended in 10 mL Histopaque solution (5:11 Histopaque-1077 (Sigma-Aldrich; 10771) and 6:11 Histopaque-1119 (Sigma-Aldrich; 11191)). The Histopaque solution was centrifuged for 20 minutes at 380 x g at RT (Acceleration = 8, break = 2). The islet-containing supernatant was filtered through a 400 μm filter (pluriStrainer; 43-50400-03), followed by a 70 μm filter (Fisherbrand; 11597522). Islets were hand-picked twice and contained in RPMI 1640+ Glutamax™ medium (Gibco; 61870044) supplemented with 5% heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Lonza; DE17-602E).We adapted the 10x Genomics protocol (CG000375, Demonstrated Protocol_NucleiIsolationComplexSample_ATAC_GEX_Sequencing_RevB) for nuclei isolation from islets. Freshly isolated islets were centrifuged for 5 minutes at 400 x g at 4°C. The media-supernatant was removed, leaving ~20 µL media on top of the islet pellet. To release the nuclei, 100 μL nuclei preparation buffer (NBP) (10 mM HEPES pH 7.5 (Sigma; H3375), 1.5 mM MgCl2 (Sigma; M8266), 40 mM KCl (Sigma; P9541), 250 mM sucrose (Sigma; S0389), 0.5 mM Spermidine (Sigma; S2626), 1% BSA (Merck; M0314L), 0.1% IGEPAL CA-360 (Sigma; I8896), 0.2 mM DTT (NEB; B1034A), and 1 U/μL RNase inhibitor (NEB; M0314) dissolved in DEPC water) was added, and the pellet was homogenized 20 times using the Biomasher II Disposable Micro Tissue Homogenizer (EOG-sterilized) (NIP; 320103). Then, 1000 μL NBP was added, and the solution was incubated on ice for 5 minutes. The nuclei suspension was passed through a 70 μm tip strainer (Merck; BAH136800070-50EA) into a 1.5 mL tube. The biomasher tube was washed with 500 μL NPB and passed through the tip strainer into the same tube. The nuclei suspension was centrifuged at 500 x g for 5 minutes at 4°C, and the supernatant was removed until ~50 μL remained. The nuclei suspension was washed with 1000 μL 2% BSA in 1X PBS + 1U/μL RNase inhibitor, incubated on ice for 5 minutes, then mixed to resuspend the nuclei pellet. The suspension was centrifuged again, and the supernatant was removed. To permeabilize the nuclei membrane, the nuclei were incubated in 100 μL 0.1X lysis buffer (10 mM Tris-HCl pH 7.5 (VWR; 103156X), 3 mM MgCl2, 10 mM NaCl (Sigma; S3014), 0.01% Tween-20 (Sigma; P1379), 0.01% IGEPAL CA-630, 1% BSA, 0.001% Digitonin (ThermoFisher; BN2006), 1 mM DTT, 1U/μL RNase inhibitor dissolved in DEPC water) for 2 minutes, then diluted with 1 mL wash buffer (10 mM Tris-HCl pH 7.5, 3 mM MgCl2, 10 mM NaCl, 0.1% Tween-20, 0.1% BSA, 1 mM DTT, 1U/μL RNase inhibitor dissolved in DEPC water). The solution was mixed and centrifuged, and the supernatant was removed. The nuclei pellet was resuspended in 60 μL 1X nuclei buffer (10x Genomics), 1 mM DTT, and 1 U/μL RNase inhibitor dissolved in DEPC water. Nuclei aggregates were removed by passing the solution through a 40 μm tip strainer (Merck; BAH136800040-50EA), and the suspension was centrifuged again. The supernatant was removed until 7 μL nuclei buffer remained. Isolated nuclei were prepared for transposition and library generation using the following 10x Genomics kits: Chromium Next GEM Single Cell Multiome GEM Kit A, 16 rxns (PN-1000232), Chromium Next GEM Single Cell Multiome Amp Kit A, 16 rxns (PN-1000233), Chromium Next GEM Single Cell Multiome ATAC Kit A, 16 rxns (PN-1000280), and Library Construction Kit, 16 rxns (PN-1000190). The samples were prepared in accordance with the manufacturer's instructions (CG000338, Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User guide). Libraries were sequenced on the Illumina NovaSeq 6000 System (Illumina, 20012850), with the following read lengths (Read1 + Index1 + Index2 + Read2): RNA: 28 + 10 + 10 + 90, and ATAC  50 + 8 + 24 + 50