title: GSM8854301: snMO, hippocampus, P5, ATAC; Mus musculus; ATAC-seq
library_construction_protocol: Nuclei preparation from mouse hippocampal tissue was done using sucrose gradient protocol. Briefly, up to 50 mg of hippocampal tissue was homogenized using 1 ml of Homogenization Lysis Buffer (HLB) (1X salt-Tris buffer: 146 mM NaCl, 10 mM Tris 7.5, 1 mM CaCl2, 21 mM MgCl2; 0.03 % v/v Tween-20, Sigma Aldrich; 0.01% v/v NP40, Sigma Aldrich; 1% BSA, Thermo Fisher; 25 mM KCl, Thermo Fisher; 250 mM Sucrose, Sigma Aldrich; 1mM DTT, Thermo Fisher; 1X Protease Inhibitor, Sigma Aldrich; 1 U/ul RNAse Inhibitor, Promega). The tissue was homogenized with 10X strokes with pestle A and 10X strokes with pestle B. The homogenized mixture was incubated in HLB for 5 min. During the incubation, the homogenized sample was passed through 70 µm nylon mesh. The nuclei were pelleted by centrifugation at 500xg for 5 min. The nuclei pellet was washed and resuspended with 520 µl of Wash Buffer 1 (1X salt-Tris buffer; 1% BSA, Thermo Fisher; 25 mM KCl, Thermo Fisher; 250 mM Sucrose, Sigma Aldrich; 1mM DTT, Thermo Fisher; 1X Protease Inhibitor, Sigma Aldrich; 1 U/µl RNAse Inhibitor, Promega). The nuclei suspension was then mixed with equal volume of Gradient Medium (1 mM CaCl2, Thermo Fisher; Optiprep 50% v/v, Sigma Aldrich; 5 mM MgCl2, Thermo Fisher; 10 mM Tris 7.5, ThermoFisher; 75 mM Sucrose, Sigma Aldrich; 1mM DTT, Thermo Fisher; 1X Protease Inhibitor, Sigma Aldrich; 0.5 U/µl RNAse Inhibitor, Promega). This was thoroughly mixed, and the mix was layered upon 948 µl of 29% density cushion (Optiprep 29% v/v, Sigma Aldrich; 72.5 mM KCl, Thermo Fisher; 14.5 mM MgCl2, Thermo Fisher; 29 mM Tris 8, Thermo Fisher; 121 mM Sucrose, Sigma Aldrich; 0.25 U/µl RNAse Inhibitor, Promega). The layered nuclei suspension was subjected to centrifugation at 9000xg for 20 min at 4 °C. After the centrifugation, the debris was removed from the top of the suspension and if excess debris was present, this could be scooped out with a 1000 µl pipette tip. The supernatant was aspirated out gradually using a glass Pasteur pipette until about 75 µl of the supernatant was left. Equal volume of 0.2X Lysis Buffer (20 mM Tris 7.5, ThermoFisher; 20 mM NaCl, ThermoFisher; 6mM MgCl2, ThermoFisher; 0.02% Tween 20, Sigma Aldrich; 0.02% NP40, Sigma Aldrich; 0.002% Digitonin, Promega; 2% BSA, Thermo Fisher; 2mM DTT, ThermoFisher; 2U/µl Protector RNAse Inhibitor, Sigma Aldrich) was added and the nuclei were resuspended and incubated for 2 min on wet ice. After the incubation the lysis was quenched by adding 1 ml of Wash Buffer 2 (10 mM Tris 7.5, ThermoFisher; 10 mM NaCl, ThermoFisher; 3 mM MgCl2, ThermoFisher; 0.01% Tween 20, Sigma Aldrich; 1% BSA, Thermo Fisher; 1 mM DTT, ThermoFisher; 1 U/µl Protector RNAse Inhibitor, Sigma Aldrich) and the nuclei suspension was resuspended. The nuclei were pelleted by centrifugation at 500xg for 5 min. The supernatant was discarded until about 20 µl of the supernatant was left in the Eppendorf. Removal was done carefully without disturbing the nuclei pellet. Nuclei pellet was resuspended with 20 µl of 2X Nuclei Buffer (2X Nuclei buffer, 10X Genomics, PN-2000153; 1 mM DTT, ThermoFisher; 1U/µl Protector RNAse Inhibitor, Sigma Aldrich). The nuclei count and the viability of the samples were accessed using LUNA dual florescence cell counter (Logos Biosystems) and a targeted cell recovery of 10000 cells was aimed for each of the samples. Library preparations for 10X Genomics single cell Multiome were performed using 10X Genomics Chromium NextGEM Single cell Multiome ATAC+Gene Expression chemistry (10X Genomics, Pleasanton, CA, USA). Transposition of nuclei and the rest of the 10X Genomics Multiome library preparation was performed as per the protocol recommendations (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide; CG000338 Rev G). At the different check points the library quality was accessed using Qubit (ThermoFisher) and Bioanalyzer (Agilent). 10X libraries were sequenced either on Illumina platform or on MGISEQ-2000 sequencing platform (MGI Hong Kong SAR sequencing facility). Before being able to sequence on the MGI platforms, the 10X libraries need to undergo a conversion step using MGIEasy Universal DNA Library Prep kit. Briefly, the final 10X sequencing libraires were circularized using the splint-ligation step and the circularized libraries were converted to single-stranded DNA copies. DNA nanoballs were prepared from the circularized ssDNA using Rolling Circle Amplification (RCA). DNB libraries generated were then flown through the patterned flow cell of the MGISEQ-2000RS High-Throughput Sequencing kit. For a targeted sequencing saturation of 50-60%, sequencing was performed at a depth of 30,000 - 60,000 reads per cell for the RNA and ATAC libraries. For RNA libraries sequencing recipe of paired end read of 28 bps (read 1), 100 bps (read 2) and 10 bps (index 1) and 10 bps (index 2) was used for sequencing. For ATAC libraries sequencing recipe of paired end read of 50 bps (read 1), 49 bps (read 2) and 24 bps (index i5) and 8 bps (index i7) were used for sequencing.