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| Status |
Public on Nov 02, 2023 |
| Title |
9H,rep2 |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain
tissue region: HPF
strain: C57BL/6J
number of_animals_pooled: 10
Sex: Male
age: 8-9 weeks old
library type: ATAC-seq
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Adult C57BL/6J male mice were purchased from Jackson Laboratories. Brains were extracted from 56-63 day old mice and sectioned into 600 µm coronal sections along the anterior-posterior axis in ice-cold dissection media. Specific brain regions were dissected according to the Allen Brain Reference Atlas (Wang, Q. et al. The Allen Mouse Brain Common Coordinate Framework: A 3D Reference Atlas. Cell (2020). https://doi.org:10.1016/j.cell.2020.04.007). For each region, dissected brain tissues were pooled from 2-31 male C57BL/6J mice to obtain enough nuclei for single nucleus ATAC-seq for each biological replica, and two biological replicas were performed.
Brain nuclei were pelleted with a swinging bucket centrifuge (500g, 5 min, 4 °C; 5920R, Eppendorf). Nuclei pellets were resuspended in 1 ml nuclei permeabilization buffer (5% BSA, 0.2% IGEPAL-CA630, 1 mM DTT and cOmpleteTM, EDTA-free protease inhibitor cocktail (Roche) in PBS) and pelleted again (500g, 5 min, 4 °C; 5920R, Eppendorf). Nuclei were resuspended in 500 μl high-salt tagmentation buffer (36.3 mM Tris-acetate (pH 7.8), 72.6 mM potassium-acetate, 11 mM Mg-acetate, 17.6% dimethylformamide) and counted using a haemocytometer. Concentration was adjusted to 1,000–4,500 nuclei per 9 μl, and 1,000–4,500 nuclei were dispensed into each well of a 96-well plate. For tagmentation, 1 μl barcoded Tn5 transposomes26 were added using a BenchSmart 96 (Mettler Toledo), mixed five times, and incubated for 60 min at 37 °C with shaking (500 rpm). To inhibit the Tn5 reaction, 10 μl of 40 mM EDTA was added to each well with a BenchSmart 96 (Mettler Toledo) and the plate was incubated at 37 °C for 15 min with shaking (500 rpm). Next, 20 μl 2× sort buffer (2% BSA, 2 mM EDTA in PBS) were added using a BenchSmart 96 (Mettler Toledo). All wells were combined into a FACS tube and stained with 3 μM Draq7 (Cell Signaling). Using a SH800 (Sony), 20 nuclei were sorted per well into eight 96-well plates (total of 768 wells, 30,720 nuclei total, 15,360 nuclei per sample) containing 10.5 μl EB (25 pmol primer i7, 25 pmol primer i5, 200 ng BSA (Sigma). If processing two samples per day, tagmentation was performed with different sets of barcodes in separate 96 well plates. After tagmentation nuclei from individual plates were pooled together. Preparation of sort plates and all downstream pipetting steps were performed on a Biomek i7 Automated Workstation (Beckman Coulter). After the addition of 1 μl 0.2% SDS, samples were incubated at 55 °C for 7 min with shaking (500 rpm). Then, 1 μl 12.5% Triton X-100 was added to each well to quench the SDS. Next, 12.5 μl NEBNext High-Fidelity 2 × PCR Master Mix (NEB) were added and samples were amplified by PCR (72 °C 5 min, 98 °C 30 s, (98 °C 10 s, 63 °C 30 s, 72 °C 60 s) × 11 or 12 cycles, held at 12 °C). After PCR, all wells were combined. Libraries were purified according to the MinElute PCR Purification Kit manual (Qiagen) using a vacuum manifold (QIAvac 24 plus, Qiagen) and size selection was performed with SPRI Beads (Beckmann Coulter, 0.55x and 1.5x). Libraries were purified one more time with SPRI Beads (Beckmann Coulter, 1.5x). Libraries were quantified using a Qubit fluorimeter (Life Technologies) and the nucleosomal pattern was verified using a Tapestation (High Sensitivity D1000, Agilent). Libraries generated with indexing version 1 were sequenced on a HiSeq2500 sequencer (Illumina) using custom sequencing primers, 25% spike-in library and following read lengths: 50 + 43 + 37 + 50 (Read1 + Index1 + Index2 + Read2). Libraries generated with indexing version 2 were sequenced on a Libraries were sequenced on a HiSeq 4000 (Illumina) or a NovaSeq 6000 (Illumina) with these settings: 50 + 10 + 12 + 50 (Read1 + Index1 + Index2 + Read2).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Paired-end sequencing reads were demultiplexed and the cell index transferred to the read name. Sequencing reads were aligned to mm10 reference genome using bwa.
We combined the sequencing reads to fragments with the function make_fragment_file in SnapATAC2. , and for each fragment we followed the quality controls: 1) Keep only fragments with quality scores MAPQ > 30; 2) remove PCR duplicates. Reads were also sorted based on cell barcodes in read names, and shifted +4 bp for positive strand and -5 bp for negative strand to correct the 9-bp duplication induced from Tn5 transposase.
We used the function filter_cells in SnapATAC2 to calculate TSSenrichment (TSSe). Nuclei with >=1,000 uniquely mapped fragments and TSSe >= 10 were filtered for each of 234 samples by following the ENCODE ATAC-seq data standards and process pipeline(https://www.encodeproject.org/atac-seq/). We used the function filter_cells in SnapATAC2 to filter out cells.
We applied a modified version of Scrublet to remove potential doublets for every sample independently using SnapATAC2. Firstly, we used the function of add_tile_matrix to add the 500-bp genomic bin features, then used the function of select_features to filter out the features with the frequencies along the samples lower than 0.5% or higher than 99.5%. Then applied the function of scrublet in SnapATAC2 to get the doublet scores. The parameter expected_doublet_rate is set to 0.08, which is based on our previous experiment on the snATAC-seq pipeline. Barcodes with the scrublet scores larger than 0.5 will be treated as potential doublets, and then removed from our analysis.
Assembly: Genome_build: mm10 (GRCm38)
Supplementary files format and content: SnapATAC2 HDF5 matrix files (cells with the raw fragments, 500bp bmat)
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| Submission date |
Nov 01, 2023 |
| Last update date |
Nov 02, 2023 |
| Contact name |
Songpeng Zu |
| E-mail(s) |
szu@health.ucsd.edu
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| Organization name |
University of California San Diego
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| Department |
Department of Cellular and Molecular Medicine
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| Lab |
Prof. Bing Ren Lab
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| Street address |
9500 Gilman Dr
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE246791 |
Single-cell analysis of chromatin accessibility in the adult mouse brain |
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| Relations |
| BioSample |
SAMN38035270 |
| SRA |
SRX22287218 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7877008_CEMBA190219_9H_rm_dlt.h5ad.gz |
475.3 Mb |
(ftp)(http) |
H5AD |
SRA Run Selector |
| Raw data are available in SRA |
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