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| Status |
Public on Mar 14, 2024 |
| Title |
GMP_Tet2_Rep3 |
| Sample type |
SRA |
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| Source name |
Mouse bone marrow
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| Organism |
Mus musculus |
| Characteristics |
tissue: Mouse bone marrow
genotype: Tet2 KO
replicate: Rep3
overexpression: Control
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| Growth protocol |
Tet2 fl/fl mice (#017573, The Jackson Laboratory) were crossed to Mx1-Cre mice (#003556, The Jackson Laboratory). Mx1-Cre negative littermates were utilized as controls. 4 doses of 12.5 ug/g pI:pC (Amersham) were administered intraperitoneally to induce Cre activity.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell sorting was performed on FACS Aria II (BD Biosciences). Single stained and Fluorescence Minus One stained cells were used as controls. Samples were incubated with the antibody cocktail in PBS 2% FBS for 30 minutes at 4 degrees before sorting. 7-AAD Viability Staining Solution (BioLegend) was included in the sample preparation for flow cytometry to exclude dead cells. For a detailed description of tagmentation protocols and buffer formulations refer to the SureCell ATAC-Seq Library Prep Kit User Guide (17004620, Bio-Rad). Sorted cells and tagmentation related buffers were chilled on ice. Lysis was performed simultaneously with tagmentation. Washed and pelleted cells were resuspended in Whole Cell Tagmentation Mix containing 0.1% Tween-20, 0.01% Digitonin, 1x PBS supplemented with 0.1% BSA, ATAC Tagmentation Buffer and ATAC Tagmentation Enzyme (ATAC Tagmentation Buffer and Enzyme are both included in the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad)). Cells were mixed and agitated on a ThermoMixer for 30 min at 37°C. Tagmented cells were kept on ice prior to encapsulation.
Tagmented cells were loaded onto a ddSEQ Single-Cell Isolator. Single-cell ATAC-seq libraries were prepared using the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) and SureCell ddSEQ Index Kit (12009360, Bio-Rad). Bead barcoding and sample indexing were performed in a C1000 Touch™ Thermal cycler with a 96-Deep Well Reaction Module: 37°C for 30 min, 85°C for 10 min, 72°C for 5 min, 98°C for 30 sec, 8 cycles of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec, and a single 72°C extension for 5 min to finish. Emulsions were broken and products cleaned up using Ampure XP beads. Barcoded amplicons were further amplified using a C1000 Touch™ Thermal cycler with a 96-Deep Well Reaction Module: 98°C for 30 sec, 6-9 cycles (cycle number depending on the cell input, Section 4 Table 3 of the User Guide) of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec, and a single 72°C extension for 5 min to finish. PCR products were purified using Ampure XP beads and quantified on an Agilent Bioanalyzer using the High-Sensitivity DNA kit. Libraries were loaded at 1.5 pM on a NextSeq 550 using the NextSeq High Output Kit and sequencing was performed using the following read protocol: Read 1 118 cycles, i7 index read 8 cycles, and Read 2 40 cycles. A custom sequencing primer is required for Read 1 (16005986, Bio-Rad; included in the kit).
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| Library strategy |
ATAC-seq |
| Library source |
genomic single cell |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Data processing |
Per-read bead barcodes were parsed and trimmed using UMI-TOOLs (https://github.com/CGATOxford/UMI-tools). Constitutive elements of the bead barcodes were assigned to the closest known sequence allowing for up to 1 mismatch per 6-mer or 7-mer (mean >99% parsing efficiency across experiments).
Paired-end reads were aligned to mm10 using BWA (http://bio-bwa.sourceforge.net/).
We used bead-based ATAC-seq processing (BAP) (https://github.com/caleblareau/bap), to identify systematic biases (i.e. reads aligning to an inordinately large number of barcodes), barcode-aware deduplicate reads, and perform merging of multiple bead barcode instances associated with the same cell. Barcode merging is necessary due to the nature of the BioRad SureCell scATAC-seq procedure used in this study, which enables multiple beads per droplet. BAP uses as input an alignment (.bam) file for a given experiment with a bead barcode identifier indicated by a SAM tag.
Genome-wide chromatin accessibility peaks were called using MACS v2 (MACS2) on the merged aligned scATAC-seq reads per condition, generating a list of peak summit calls per condition. To generate a non-overlapping set of peaks, we first extended summits of each condition to 800-bp windows (±400 bp). We combined these 800-bp peaks, ranked them by their summit significance value and retained specific non-overlapping peaks on the basis of this ordering. We further added to the peak list all non-overlapping peaks from the ImmGen ATAC-seq atlas, after also extending the ImmGen peaks to 800-bp windows (https://sharehost.hms.harvard.edu/immgen/ImmGenATAC18_AllOCRsInfo.csv). This resulted in a filtered list of disjoint peaks (n=297,361), which were finally resized to 301 bp (i.e. ±150 bp from each peak summit) and used for all downstream analyses.
A matrix of reads in peaks counts for single cells was obtained by counting the number of uniquely aligned fragments (with coordinates offset +4/-5 bp for the +/- strand to adjust for Tn5 integration) overlapping the determined peak window genomic ranges, grouped by cell barcode ID, done using custom R code.
Assembly: mm10
Supplementary files format and content: Peak file (BED) contains chromosome, start and end coordinates for determined chromatin accessibility peak window ranges. cellMeta file contains single cell barcode information for the final set of filtered scATAC-seq cells used in the analysis. Fragments.tsv.gz files contain all Tn5-corrected coordinates for aligned fragments generated using BAP, which can be used to overlap with peaks to get counts of Tn5 insertions falling within peaks for each cell barcode. Raw_counts.mtx file contains the peaks x cells count matrix and is indexed by gene (row), cell (column), and count.
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| Submission date |
Nov 16, 2023 |
| Last update date |
Mar 14, 2024 |
| Contact name |
Fabiana Duarte |
| E-mail(s) |
fabiana_duarte@g.harvard.edu
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| Organization name |
Harvard University
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| Department |
HSCRB
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| Lab |
Buenrostro lab
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| Street address |
7 Divinity Avenue
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| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE227314 |
Cell of origin epigenetic priming determines susceptibility to Tet2 oncogenic mutation [scATAC-seq] |
| GSE247970 |
Cell of origin epigenetic priming determines susceptibility to Tet2 oncogenic mutation |
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| Relations |
| BioSample |
SAMN38283055 |
| SRA |
SRX22545608 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7903898_A0_gmp.fragments.tsv.gz |
298.6 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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