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| Status |
Public on Jul 24, 2024 |
| Title |
Replicate 4, dialout library |
| Sample type |
SRA |
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| Source name |
HEK293T
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei were prepared according to 10X Genomics’ Nuclear Isolation for Single Cell ATAC Sequencing protocol with the following notes. IGEPAL CA-630 was used for the lysis buffer a 10% solution of which was prepared fresh on the day of nuclear isolation using 100% stock (Sigma i8896). After trypsinization and pelleting, cells were washed twice with ice cold PBS + 0.04% BSA before following the remainder of the nuclear isolation protocol. Lysis occurred for 4 minutes which previously was found to be appropriate time for cytoplasmic removal while retaining nuclear membrane architecture by DIC microscopy. Cells were resuspended in diluted nuclei buffer and triplicates were counted before proceeding with Spear-ATAC sequencing as previously published.
Library construction was performed according to 10X Genomics scATAC-seq v2 Chemistry and Spear-ATAC with the following modifications and notes. 10,000 nuclei were targeted for capture per 10X chip lane. Spear-ATAC included the following changes to the 10X scATAC library preparation protocol: the 1.2 uL per reaction of 50 uM oSP1735 oligonucleotide is spiked into the GEM generation master mix and the in-GEM PCR was increased from 12 to 15 cycles. Single-cell ATAC library fragment size distribution was analyzed by Tapestation prior to preparing enriched dial-out libraries for perturbation genotyping. The dial-out libraries were again prepared according to the Spear-ATAC protocol with the following changes: linear PCR with biotinylated oSP2053 was increased to 30 total cycles and the exponential PCR with oSP1594 and indexed P7 primers was increased to 18 total cycles. The indices on the P7 primers were increased from 8 bases to 10 bases.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
For scATAC/Spear-ATAC libraries: Sequencing data was converted to fastq files using cellranger-atac mkfastq (10x Genomics, version 2.1.0). Reads were then aligned to the hg38 reference genome and quantified using cellranger-atac count. The current version of Cell Ranger can be accessed here:https://support.10xgenomics.com/single-cell-atac/software/downloads/latest
For dialout libraries: To filter for reads that contain the correct structure of amplicon, we grepped for 4 constants sequences that flanked the barcodes: “AAATCCAAGC”, “CCAGAGCATG”, “CAAGGTGGTT”, and “ATACTGATTC”. . We then matched each of the supposed hardcoded barcodes to one of the known hardcoded barcodes from the list that we intended to synthesize by identifying a closest match using a maximum restricted Damerau-Levenshtein distance of 3. We then generated a list of unique hardcoded barcode – random barcode pairs. We restricted that list to pairs to those that were seen more than once. Finally, we required that, for a given random barcode, more than 95% of the reads had to match to the same hardcoded barcode.
Assembly: hg38
Supplementary files format and content: Tab separated values
Library strategy: Spear-ATAC
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| Submission date |
Sep 19, 2023 |
| Last update date |
Jul 24, 2024 |
| Contact name |
Max Frenkel |
| E-mail(s) |
mfrenkel@wisc.edu
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| Organization name |
UW Madison
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| Street address |
433 babcock drive
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| City |
Madison |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (1) |
| GSE243553 |
Large-scale discovery of chromatin dysregulation induced by oncofusions and other protein-coding variants |
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| Relations |
| BioSample |
SAMN37458213 |
| SRA |
SRX21825931 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7790874_Sample4_merged_associations.csv.gz |
100.6 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
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