|
| Status |
Public on Apr 30, 2021 |
| Title |
scATAC-aSP71_KPL_Primary_13C |
| Sample type |
SRA |
| |
|
| Source name |
Mouse lung adenocarcinoma primary tumors and metastases
|
| Organism |
Mus musculus |
| Characteristics |
tissue/cell type: lung cancer
source: mouse derived
growth media: n/a
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Up to 50,000 FACS-isolated lung adenocarcinoma cells were viably cryopreserved in BAMBANKER freezing media (Wako Chemicals 302-14681) so that scATAC-seq could be performed on all cells at the same time. Cells were pelleted in a fixed-angle rotor at 300 RCF for 5 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in 100 uL of ice-cold lysis buffer (RSB containing 0.1% Tween-20, 0.1% NP-40, and 0.01% digitonin) and incubated on ice for 3 minutes. To dilute lysis, 1 mL of chilled RSB-T was added to each tube and the cells were pelleted as before. The supernatant was removed and the pelleted nuclei were resuspended in Diluted Nuclei Buffer (10x Genomics). The nuclei stock concentration was determined for each cell line using Trypan Blue and a total of 5,000 nuclei from each cell line were pooled together and loaded into the 10x Genomics scATAC-seq (v1) transposition reaction.
scATAC-seq library preparation was performed as recommended by the manufacturer (10x Genomics scATAC-seq (v1)).
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
Single Cell ATAC-seq
processed data file:
Save_LKB1_ArchRProject.zip
scATAC-LKB1-PeakMatrix-Summarized-Experiment.rds
scATAC-LKB1-GeneScoreMatrix-Summarized-Experiment.rds
scATAC-LKB1-ChromVAR-Summarized-Experiment.rds
|
| Data processing |
ATAC-seq:
Fastq reads were trimmed using cutadapt and then aligned to the genome with bowtie 2 and then filtered for high quality read paired alignment.
Mitochondrial and picard identified pcr duplicates were then discarded.
Macs2 was then used to identify peak regions that were reproducible across replicate samples and then to create a sample by peak matrix.
Bulk RNA-seq:
Fastq reads were trimmed using cutadapt and then pseudo aligned to the genome with Kallisto.
Counts were then compiled from each sample to build a sample by gene counts matrix.
Single Cell ATAC-seq:
The Cell Ranger ATAC Software Suite (version 1.2.0) was used for processing sequencing information and single cell barcodes
Read pairs were aligned and deduplicated to yield fragment intervals whose ends signify accessibiltiy sites along the genome where the Tn5 transposase made cuts
Regions of enriched accessibility, i.e. peaks were identified with MACS2
Barcodes likely associated with whole cells were identified by analyzing the number of fragments (1000) mapped near TSS regions (enriched over background) and a peak-barcode matrix is defined
CRISPR Screen:
Fastq reads were aligned to sgRNA barcodes using casTLE (implementing bowtie).
Counts were then compiled from each sample across both screen halves and normalized.
The counts matrix was then used as input to MaGeCK for downstream analysis.
Genome_build: mm10/hg38
Supplementary_files_format_and_content:
Processed ATAC-seq/scATAC-seq data is provided as an R SummarizedExperiment object. This object contains counts alongth with row and column metadata corresponding to the individual cells and peak/motif/gene chromatin accessibility. Fragments file contain Tn5 corrected cut sites per sample or cell.
Processed RNA-seq data is provided in an xlsx file containing counts/tpm for each sample per gene. Additionally, Kallisto generated transcript abundances per sample are provided.
Processed CRISPR Screen data using casTLE (bowtie) is provided as a counts table for each sgRNA count sample per gene and a table of results from MAGeCK analysis.
*insertion.bw: Sample Bigwig file of chromatin accessibilty
*Fragments.tsv.gz: Sample Fragments Files containing Tn5 cut sites of chromatin accessibility
*chromVAR-Summarized-Experiment.rds: Matrix summarized experiment file (R object) containing samples by TF accessibility.
*Peak-Summarized-Experiment.rds: Matrix summarized experiment file (R object) containing samples by peak accessibility.
*abundance.tsv: Sample transcript abundances from Kallisto.
bRNA-Matrices-Initial-LKB1-Resoration.xlsx: Matrix excel file containing transcript abundances and counts for each sample with descriptions.
bRNA-Matrices-Mouse-Tumor-Metastases-Matrices.xlsx: Matrix excel file containing transcript abundances and counts for each sample with descriptions.
Save_LKB1_ArchRProject.zip: Saved ArchRProject from ArchR for scATAC-seq mouse data.
*singlecell.csv: 10x Genomics Single Cell QC statistics file.
*fragments.tsv.gz: Sample Fragments Files containing Tn5 cut sites per cell of chromatin accessibility
*chromVAR-Summarized-Experiment.rds: Matrix summarized experiment file (R object) containing cells by TF accessibility.
scATAC-LKB1-GeneScoreMatrix-Summarized-Experiment.rds: Matrix summarized experiment file (R object) containing cells by gene accessibility.
scATAC-LKB1-PeakMatrix-Summarized-Experiment.rds: Matrix summarized experiment file (R object) containing cells by peak accessibility.
Screen-Counts-s1*counts.csv: Counts file from casTLE for each sample of CRISPR screen half 1.
Screen-Counts-s1_s2*counts.csv: Counts file from casTLE for each sample of CRISPR screen half 2. (Repeat of sample from half 1)
Screen-Counts-s2*counts.csv: Counts file from casTLE for each sample of CRISPR screen half 2.
Screen-Matrices-Combined-sgRNA-Counts.tsv: Combined Matrix file of all samples for CRISPR screen.
Screen-Matrices-Mageck-Results-Gene-Summary.txt: Results file from MaGeCK analysis of CRISPR screen data.
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| |
|
| Submission date |
Feb 23, 2021 |
| Last update date |
Apr 30, 2021 |
| Contact name |
Jeffrey Michael Granja |
| E-mail(s) |
[email protected]
|
| Organization name |
Stanford University
|
| Department |
Genetics
|
| Lab |
Greenleaf
|
| Street address |
279 Campus Drive W
|
| City |
STANFORD |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL21626 |
| Series (1) |
| GSE167381 |
LKB1 inactivation initiates a chromatin remodeling cascade to drive metastatic progression |
|
| Relations |
| BioSample |
SAMN18042292 |
| SRA |
SRX10159761 |
