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| Status |
Public on Dec 22, 2020 |
| Title |
sRNA K562i ZSWIM8 knockdown gRNA A rep2 |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
cell type: K562 expressing inducible KRAB-dCas9 (K562i)
grna: ZSWIM8 gRNA A
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were pelleted by centrifugation and washed with PBS. RNA was harvested using TRI-Reagent.
Small-RNA sequencing libraries were prepared as described (Kingston and Bartel, 2019). A step-by-step protocol is available at http://bartellab.wi.mit.edu/protocols.html. Libraries were prepared from 5 ug total RNA extracted from K562i cells.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Lentiviral transduction of gRNA
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| Data processing |
Standard base calls were made using the Illumina HiSeq platform protocol.
Adapter sequences were trimmed using cutadapt (Martin, 2011) and filtered using fastq_quality_filter (FastX Toolkit; http://hannonlab.cshl.edu/fastx_toolkit/) with the parameters ‘-q 30 –p 100’ to ensure that all bases had an accuracy of 99.9%.
Counting of miRNAs was performed by string-matching the first 19 nt of each read to a dictionary derived from mature miRNA sequences. These dictionaries were generated from sets of mature miRNA sequences downloaded from TargetScan7 (Agarwal et. al, 2015) in the case of human, mouse, and fly miRNAs, or as reported (Jan et. al, 2011) in the case of worm miRNAs. These sequences were filtered to retain only those for which the first 19 nt were distinct. This matching method had the advantage of reducing the ambiguity in assigning miRNA isoforms, which exhibited heterogeneity at their 3′ ends, to a single mature miRNA.
Count files were merged to generate tables of counts for each cell line, organized by the gRNA expressed for knockdown/knockout.
Differential expression and significance levels were determined using DESeq2 (1.18.1) (Love et. al, 2014) without the lfcShrink() function.
Genome_build: TargetScanHuman release 7.2 or TargetScanMouse release 7.2
Supplementary_files_format_and_content: Tab-delimited text files generated by DESeq2 contain normalized counts for each library, log2-fold changes, adjusted p-values, and other output.
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| Submission date |
Oct 28, 2020 |
| Last update date |
Jan 06, 2021 |
| Contact name |
Charlie Y. Shi |
| E-mail(s) |
[email protected]
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| Organization name |
Whitehead Institute / MIT
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| Department |
Biology
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| Lab |
Bartel Lab
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| Street address |
455 Main St.
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE160304 |
The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation |
| GSE163393 |
The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation [dataset 1] |
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| Relations |
| BioSample |
SAMN16581100 |
| Supplementary data files not provided |
| Raw data are available in SRA |
| Processed data are available on Series record |
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