ZT12_2
Mus musculus
Protocols: Mouse left ventricular biopsies were harvested at ZT0 and at ZT12 from C57BL/6J mice acclimatised to a 12 h light:12 h dark cycle. Frozen left ventricle samples were added directly to 1 ml cold lysis buffer (containing 0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 2 mM EDTA pH 8, 0.5 mM EGTA, 10 mM Tris-HCl pH 8, 1 mM DTT, 1x protease inhibitor cocktail, 80 U/ml Rnasin Plus and 0.2% Triton X-100) and quickly minced into small pieces using microdissection scissors in a petri dish on ice. In a 1 ml dounce homogeniser, 18 x loose strokes were immediately followed by 3-4 x tight strokes. To eliminate large debris, the suspension was passed over 2 layers of sterile cotton gauze prior to filtration with a 100 mM pluriStrainer (pluriSelect) and a 40 mM Flowmi filter (Sigma). Filtered suspensions were centrifuged at 1000 x g for 8 min at 4°C to pellet the nuclei. The nuclei pellet was suspended in 200 ml ATAC-RSB+Tween (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) and a 5 ml aliquot added in a 1:1 ratio with trypan blue for counting using a manual haemocytometer and light microscope. To isolate cardiomyocyte nuclei, suspensions were pelleted (1000 x g for 8 min at 4°C) in ATAC-RSB-Tween and then resuspended in 500 ml staining buffer (PBS + 2.5% bovine serum albumin) containing rabbit anti-PCM1 primary antibody (1:400, Atlas Antibodies #HPA023374). Tubes were agitated on a rocker at 4°C for 1h. Nuclei were pelleted (1000 x g for 8 min at 4°C) again prior to resuspension in staining buffer containing goat anti-rabbit Cy5 secondary antibody (1:1000, Abcam #ab97077) and DAPI (1:10,000). Tubes were agitated on a rocker at 4°C for 1h. Subsequently, nuclei were pelleted again and resuspended in ATAC-RSB-Tween for imaging flow cytometry or fluorescence-activated nuclei sorting. Sorting of single nuclei was performed on a BD Influx Cell Sorter (BD Biosciences) using a 100mm diameter nozzle and 20 PSI. System alignment and drop delay settings were established using Cytometry Set Up and Tracking (CS&T) beads (BD Biosciences) and AccuDrop beads (BD Biosciences) respectively prior to sorting. Nuclear fraction samples were stained with DAPI and Cy5 before running on the cell sorter. Nuclei concentration was adjusted to run samples at 5,000 events per second and sorted nuclei were collected into individual 1.5 ml polypropylene tubes containing ATAC-RSB-Tween. ATAC-seq library preparation and sequencing. Nuclei from the \"sample collection protocol\" were added directly to the library preparation protocol, see \"nucleic acid library construction protocol\". ATAC-seq libraries were prepared using an Omni-ATAC-Seq protocol. 50,000 nuclei were pelleted by centrifugation at 1000 x g for 8 min at 4°C in ATAC-RSB-Tween. The supernatant was discarded ensuring not to disturb the pellet. Tn5 transposition mixtures were prepared using the Illumina Tagment DNA TDE1 Enzyme and Buffer kit (#20034197) to a final volume of 50 ml containing 25 ml 2x TD buffer, 2.5 ml TDE1 transposase, 16.5 ml PBS, 0.5 ml 0.5% digitonin, 0.5 ml 10% Tween-20 and 5 ml nuclease-free H2O. The transposition mixture was added to the nuclei pellet, pipetting up and down 10 x to mix. Reactions were incubated at 37°C for 30 min at 1000 rpm in an Eppendorf Thermomixer. Following transposition, DNA was cleaned using the Zymo DNA Clean and Concentrator-5 kit (#D4014), eluting the purified DNA fragments in a final volume of 20 ml. Following the transposase reaction, the DNA fragments were PCR amplified with combined dual indexing primers (Integrated DNA Technologies) to produce libraries for sequencing. Library pre-amplification was performed in reactions containing 20 ml DNA, 25 ml NEBNext High-Fidelity 2 ´ PCR master mix (New England BioLabs, #M0541S) and 6.25 ml of primer mix. Thermal cycling was performed at 72°C for 5 min, 98°C for 30 sec, then 5 cycles of 98°C for 10 sec, 63°C for 30 sec and 72°C for 1 min. qPCR was used to determine the number of additional amplification cycles required for each sample in 15 ml reactions: 5 ml pre-amplified mixture, 5 ml NEBNext High-Fidelity 2 ´ PCR master mix, 1.25 ml primer mix, 0.24 ml 25´ SYBR Green I (Invitrogen, #S7563) and 3.51 ml nuclease-free H2O. Thermal cycling was performed on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems): 98°C for 30 sec. then 20 cycles of 98°C for 10 sec, 63°C for 30 sec and 72°C for 1 min. Data were collected using QuantStudio Software V1.3 (Applied Biosystems). Relative fluorescence (Rn) values were exported and analysed in Microsoft Excel to determine additional PCR cycles, by determining the number of cycles required for the amplification to reach the exponential phase (1/3 of the maximum-minimum Rn). Additional PCR amplification cycles were performed in a thermal cycler. Following library amplification, a double-sided fragment size selection was performed using Ampure XP beads (Beckman Coulter, #A63880) using a final 1.8x bead buffer:sample ratio to remove primer dimers and fragments >1,000 bp. Library fragment size distribution was assessed by Agilent TapeStation 2200 using Agilent D1000 ScreenTape (#5067-5582) and reagents (#5067-5583).
strain
C57BL/6J
individual
4
organism
Mus musculus
organism
Mus musculus
collection date
not collected
sex
male
scientific_name
Mus musculus
common name
house mouse
organism part
heart left ventricle
developmental stage
adult
genotype
wild type genotype
isolate
not applicable
ENA-FIRST-PUBLIC
2024-02-29T12:32:26Z
ENA-LAST-UPDATE
2024-02-29T12:32:26Z
age
8 to 10
geographic location (country and/or sea)
not collected
ENA-FIRST-PUBLIC
2024-02-29
ENA-LAST-UPDATE
2024-02-29