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| Status |
Public on Dec 05, 2016 |
| Title |
wt 24h rep2 (siRNA-Seq) |
| Sample type |
SRA |
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| Source name |
Cells
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: wt (975 h+)
treatment: N starvation
time: 24h after -N
ip antibody: FLAG Magnetic bead (Medna Bio P8101ML1)
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| Growth protocol |
Cells grown in EMM 225ug/ml adenine/histidine/leucine/lysine/uracil (OD~1) at 32C, and were washed twice with EMM -N. Cells were resuspended in EMM -N and incubated at 32C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Ago1-bound sRNAs were isolated and sequenced using the protocol described in Holoch and Moazed (Holoch and Moazed, 2015b). Briefly, cell pellets from freshly grown proliferative and quiescent cells were frozen as droplets in liquid nitrogen. Cells were lysed by grinding in Retsch Cryomill (pre-chilled in liquid nitrogen) using Tissue Lyser II (5x 2 min at 30Hz). Lysed cells were thawed at room temperature for 10 min and then resuspended in 1ml of IP buffer (50 mM HEPES, pH 7.4, 5mM Magnesium acetate, 1mM EDTA, 1mM EGTA, 300mM Sodium acetate, 5% glycerol, 0.5% CHAPS, 1 mM PMSF and Roche complete EDTA-free protease-inhibitor cocktail) per gram of lysed cells. The lysate was clarified by centrifugation and the supernatant was incubated with pre-equilibrated anti-DYKDDDDK magnetic microbeads (Medna Bio # P8101ML1) for 4 h at 4 °C. Beads were washed 3x 1 ml lysis buffer, resuspended in 500 μl of nuclease free water, subjected to Phenol/chloroform extraction followed by ethanol precipitation. The RNA samples were resuspended in nuclease free water, electrophoresed on 17.5% Polyacrylamide/7M Urea gel and RNA species between 20-30 nucleotides were gel-extracted. Gel slices were crushed and resuspended in 0.4M sodium acetate (pH = 5.2) overnight at 4 deg C. Extracted siRNAs were ethanol precipitated and resuspended in nuclease-free water. These purified siRNAs were used for library preparation as described previously (Holoch and Moazed, 2015b).
Custom library based on Holoch and Moazed, 2015b
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Ago1-associated RNA
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| Data processing |
Remove adapter with Cutadapt
Map go S pombe genome with Bowtie2 2.1.0
Select reads that starts with "U"
Select reads between 18 and 25nt
Quantify the number of reads per transcript by HTSeq 0.6.1
Genome_build: S. pombe ASM294v2.22
Supplementary_files_format_and_content: Number of reads per transcript. Txt file
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| Submission date |
Oct 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard Joh |
| E-mail(s) |
[email protected]
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| Organization name |
Mass General Hospital
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| Department |
Center for Cancer Research
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| Street address |
149 13th St
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| City |
Charlestown |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
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| Platform ID |
GPL17225 |
| Series (2) |
| GSE89150 |
Survival in quiescence requires the euchromatic deployment of Clr4/SUV39H by Argonaute-associated small RNAs [siRNA-Seq] |
| GSE89151 |
Survival in quiescence requires the euchromatic deployment of Clr4/SUV39H by Argonaute-associated small RNAs |
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| Relations |
| BioSample |
SAMN05938982 |
| SRA |
SRX2267922 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2359778_4_htseq.txt.gz |
28.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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