GSM7852211: mouse_brain; Mus musculus; ATAC-seq
Human cortex samples from the middle frontal gyrus were sourced from the Oregon Brain Bank from a 50-year-old female of normal health status. Samples were collected by an OHSU neuropathologist, placed into a labeled cassette, and cryopreserved in an airtight container in a -80 °C freezer. The duration of time between the time of death and brain biopsy sample freezing, or post-mortem interim (PMI), was <24 hours. Mouse brain tissue was collected as discarded tissue from mice used for unrelated studies approved by the OHSU IACUC. Whole mouse brains were dissected from sacrificed C57BL/6J mice and flash-frozen in an isopentane-LN2 double-bath and stored at -80 °C. At the time of nuclei dissociation, 50 ml of nuclei isolation buffer (NIB-HEPES) was freshly prepared with final concentrations of 10 mM HEPES-KOH (Fisher Scientific, BP310-500 and Sigma Aldrich 1050121000, respectively), pH 7.2, 10 mM NaCl (Fisher Scientific S271-3), 3mM MgCl2 (Fisher Scientific AC223210010), 0.1 % (v/v) IGEPAL CA-630 (Sigma Aldrich I3021), 0.1 % (v/v) Tween-20 (Sigma-Aldrich P-7949) and diluted in PCR-grade Ultrapure distilled water (Thermo Fisher Scientific 10977015). After dilution, two tablets of Pierce™ Protease Inhibitor Mini Tablets, EDTA-free (Thermo Fisher A32955) were dissolved and suspended to prevent protease degradation during nuclei isolation. An at-bench dissection stage was set up prior to nuclei extraction. A petri dish was placed over dry ice, with fresh sterile razors pre-chilled by dry-ice embedding. 7 ml capacity Dounce homogenizers were filled with 2 ml of NIB-HEPES buffer and held on wet ice. Dounce homogenizer pestles were held in ice-cold 70% (v/v) ethanol (Decon Laboratories Inc 2701) in 15 ml tubes on ice to chill. Immediately prior to use, pestles were rinsed with chilled distilled water. For tissue dissociation, mouse and human brain samples were treated similarly. The still-frozen block of tissue was placed on the clean pre-chilled petri dish and roughly minced with the razors. Razors were then used to transport roughly 1 mg of the minced tissue into the chilled NIB-HEPES buffer within a Dounce homogenizer. Suspended samples were given 5 minutes to equilibrate to the change in salt concentration prior to douncing. Tissues were then homogenized with 5 strokes of a loose (A) pestle, another 5-minute incubation, and 5-10 strokes of a tight (B) pestle. Nuclei were transferred to a 15 ml conical tube and pelleted with a 400 r.c.f centrifugation at 4 °C in a centrifuge for 10 minutes. The supernatant was removed and pellets were resuspended in 5 ml of ATAC-PBS buffer (APB) consisting of 1X PBS (Thermo Fisher 10010) and 0.04mg/ml (f.c.) of bovine serum albumin (BSA, Sigma Aldric A2058). Samples were then filtered through a 35 µm cell strainer (Corning 352235). A 10 μl aliquot of suspended nuclei was diluted in 90 μl APB (1:10 dilution) and manually counted on a hemocytometer with Trypan Blue staining (Thermo Scientific T8154). The stock nuclei suspension was then diluted to a concentration of 2,857 nuclei/μl in APB. Dependent on experimental schema, pools of tagmented nuclei were combined to allow for the assessment of pure samples and to test index collision rates. Tagmentation plates were prepared by the combination of 1430 μl of TBS with 770 μl nuclei solution. The TBS recipe was described in “Blocking barcode-swapping”, but a different version of Digitonin (Bivision 2082-1) was used here. This solution was mixed briefly on ice. 20 μl of the mixture was placed into the 96-well iTSM plate (see “Sample multiplexing” for details). Tagmentation was performed at 37 °C for 60 minutes on a 300 r.c.f Eppendorf ThermoMixer with a lid heated to 65 °C. Following this incubation, plate temperature was brought down with a 5-minute incubation on ice to stop the reaction. Tagmented nuclei were then pooled into a single 15 ml conical tube. 5 ml of tagmentation wash buffer (TMG) was prepared consisting of a final concentration of 10 mM Tris Acetate pH 7.5 (Sigma 93352 and Sigma A6283, respectively), 5 mM MgAcetate (Sigma M5661), and 10% (v/v) glycerol (Sigma G5516), diluted in PCR grade water. 1 ml of TMG was added on top of the chilled tagmented nuclei. Nuclei were pelleted at 500 r.c.f for 10 minutes at 4 °C. Most of the supernatant was removed with care not to disturb the pellet. Then 500 μl of TMG was added to the pellet and the tube was once again spun at 500 r.c.f. for 5 minutes at 4 °C. 490 μl was removed leading to a low volume of concentrated nuclei. Loading buffer was prepared consisting of 10% (v/v) glycerol, 20 mM NaCl, 10 mM Tris-Cl pH 7.5 (Life technologies AM9855), 0.02 mM EDTA (Fisher Scientific AM9260G), 0.2 mM DTT (VWR 97061-340), and 0.2x (v/v) TB1 (Illumina Inc.). The nuclear pellet was resuspended with an additional 30 μl of loading buffer. An aliquot of 2 μl of sample was diluted 20-50X and quantified with Trypan Blue on a hemocytometer. Depending on the experiment, a 14 μl nuclei solution containing the desired amount of nuclei in the loading buffer was then combined with 1 μl of 75 μM short SBS oligo (Table S8). The 10X Chromium was then run with the custom nuclei solution as per the manufacturer's instructions (10x Document CG000209 Rev D) with the following adaptations. In step 2.4e during GEM aspiration and transfer, 100 μl GEM volume was split into two tubes, with one receiving 10 μl and the other 90 μl (henceforth referred to as 10% and 90% samples). In step 2.5.a, GEM incubation cycles were limited to 6. For Pre-PCR wash elution (Step 3.2.j) the 10% sample was eluted in 8.5 μl whereas the 90% sample was eluted in 32.5 μl. For step 3.2.n, the 10% sample had 8 μl transferred to a new strip, while the 90% sample had 32 μl transferred to a new strip. At step 4.1.b, the sample Index PCR mix was split with 11.5 μl and 46 μl being combined with the 10% and 90% samples, respectively. For step 4.1.c, 1 μl and 2 μl of a 10 μM i7 TruSeq primer was used, respectively. For step 4.1.d, 8 and 7 PCR cycles were used, respectively. Libraries were then checked for quality and quantified by Qubit DNA HS assay (Agilent Q32851) and Tapestation D5000 (Agilent 5067-5589) following the manufacturer's instructions. Libraries were then diluted and sequenced on a NextSeq 500 Mid flow cell or a NovaSeq 6000 S4 flow cell (Illumina Inc.).