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| Status |
Public on Jun 03, 2023 |
| Title |
Mock5 |
| Sample type |
SRA |
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| Source name |
Placenta
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Placenta
cell line: Primary
cell type: Trophoblast
treatment: Mock
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| Extracted molecule |
total RNA |
| Extraction protocol |
Argonaut High-throughput Sequencing UV-crosslinking and Immunoprecipitation (AGO-HITS-CLIP). Cell pellets were then subject to UV-crosslinking (25 kilograys), spun down, washed, and flash-frozen in liquid nitrogen, and stored at -80C. We then performed AGO-HITS-CLIP as described previously77,78 except for the use of photoactivatable ribonucleoside (PAR) analog thiouridine, as done in PAR-CLIP, which we did not include. Cell pellets were lysed (recipe ), DNAse (cat.) and RNAse (cat.) treated, and subject to immunoprecipitation of argonaut 2 (cat . ). Several high-salt stringent washes purified RISC-associated RNAs, proteinase (cat. ) treated, then 32P-radiolabeled, and size-selected by gel purification. These RNAs were then eluted and prepared for NGS using the TruSeq small RNA library (cat.) by ligations of the 3’ and 5’ adapters, reverse transcribed (cat.), and barcodes were added during PCR amplification of cDNA by the minimum number of PCR cycles (10-15 cycles) using a high-fidelity polymerase (cat.). TapeStation confirmed the purity of NGS libraries, and the libraries were single-end sequenced using the Illumina HiSeq 2500 platform (50-51 bp reads).
Truseq small RNA
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
AGO-HITS-CLIP
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| Data processing |
Ribonomics analyses. Reads were pre-processed and quality filtered using FastqQC (v0.11.9). Barcodes and adapters were trimmed using Trimmomatic79 (v0.33). Reads were aligned to custom human + ZIKV + hsa-miRbase transcriptome (see GRCh38.p13_and_NC_012532.1_and_hsamiRbase22.1.gtf) using STAR80 (v2.7.8). PCR duplicate reads were removed using Picard81 (v2.24.0). Unique reads were counted using HTseq82 (v0.11.1). Counts from the 5 mock and 4 infected biological replicates were used for differential expression (DE) analysis in R (v4.0.2) using DEseq283 (v3.12). AGO-HITS-CLIP peaks were called with Piranha84 (v1.2.1) or PureCLIP85 (v1.3.1). Positive or negative-sense reads-per-million-scaled (RPM) reads, and CLIP peaks were converted to bigwig files for visualization on the UCSC genome browser86 (hg38). Host miRNAs with different accumulation levels were analyzed by mirPath26 (v3, DIANA Tools) using the MicroT-CDS database25 (v5) and visualized using the Kanehisa Laboratories Pathway Viewer.
Assembly: GRCh38.p13_and_NC_012532.1_and_hsamiRbase22.1.gtf deposited to GitHub
Supplementary files format and content: htseq counts matrix
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| Submission date |
Jun 07, 2022 |
| Last update date |
Jun 03, 2023 |
| Contact name |
Michael Jochum |
| E-mail(s) |
michael.jochum@bcm.edu, michaeljochumjr@gmail.com
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| Phone |
5127698372
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| Organization name |
Baylor College of Medicine
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| Department |
Obstetrics and Gynecology
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| Lab |
Aagaard
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| Street address |
1 Baylor Plaza
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77401 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE205609 |
Zika virus co-opts miRNA networks to persist in placental microenvironments detected by spatial transcriptomics [AGO-HITS-CLIP] |
| GSE205632 |
Zika virus co-opts miRNA networks to persist in placental microenvironments detected by spatial transcriptomics |
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| Relations |
| BioSample |
SAMN28900621 |
| SRA |
SRX15624765 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6215463_Mock_3-1.txt.gz |
150.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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