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| Status |
Public on Dec 26, 2013 |
| Title |
ura4-UTRD dcr1∆ |
| Sample type |
SRA |
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| Source name |
haploid cells
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: h- ade6-M210 leu1-32 ura4-UTRD::kanMX dcr1∆::hphMX
strain: SPY 2433
molecule subtype: Ago1-associated siRNA
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| Growth protocol |
Cells were grown in minimal media lacking leucine (EMMC-LEU)
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| Extracted molecule |
total RNA |
| Extraction protocol |
To purify Ago1-associated sRNAs, cells transformed with plasmid pREP1-N3X-FLAG-Ago1 (Buker et al., 2007) were grown in 3L EMMC–Leu to a concentration of 1-2x107 cells/ml, spun down, washed twice in water, and pellets flash-frozen in liquid nitrogen. Cells were resuspended in 1 ml lysis buffer (50mM HEPES pH 7.6, 5mM MgOAc, 0.1 mM EDTA, 0.1 mM EGTA, 300 mM NaOAc, 5% glycerol, 1mM PMSF, 0.25% NP-40, plus Roche Complete Protease Inhibitor Cocktail tablet) per 1g cells in 15 mL tubes. Glass beads (Biospec, >1vol/vol cell suspension, 0.5 mm) were added and cells were disrupted in a FastPrep®-24 homogenizer (MP Biomedicals) at 6.5 meters/second for 3 times 30 s. Tubes were punctured with a 21 gage needle and the flow-through was collected in 50 ml tubes by spinning at 1000 rpm for 2 min. The flow-through was transferred to fresh tubes and spun down in a microfuge (Eppendorf 5415R) at 4300 rpm for 15 min. The supernatant was then transferred to a fresh tube. For each 1 ml supernatant, 15 ul pre-washed M2 anti-Flag resin was added and samples were incubated at 4°C for 2h. The resin was then collected by spinning at 1400 rpm for 1 min (Eppendorf 5415R), washed 3 times with lysis buffer, and once with lysis buffer without NP-40. The washed resin was resuspended in diethyl pyrocarbonate-treated water, and extracted with an equal volume of Phenol/Chloroform/Isoamylalcohol. The aqueous phase was transferred to phase lock tubes and extracted again with Phenol/Chloroform/Isoamylalcohol, precipitated by the addition of 2 volume EtOH, 1/10 volume 3M NaOAc pH 5.2, and 60 ug glycogen, and resuspended in 10 ul H2O.
Ago1-associated small RNA libraries were constructed as previously described (Halic and Moazed, 2010). Briefly, 21-24nt RNA was size-selected on a 17.5% polyacrylamide/7M urea gel and ligated to a 3’ adapter. The ligated species were size-selected on a 17.5% polyacrylamide/7M urea gel and ligated to a 5’ adapter. RNA was then reverse transcribed into cDNA and PCR-amplified in a two-step process.
sequencing of Ago1-associated small RNAs
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Sample 6
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| Data processing |
Base-calling was performing using CASAVA-1.7.0.
First, reads containing linker sequence and long polyA tracts were removed. Remaining reads were aligned using Maq (http://maq.sourceforge. net/) to the S. pombe h- genome.
Reads were then converted to an Integrated Genome Viewer (IGV)-viewable format, mapping the 3' most nucleotide of each small RNA.
The wildtype genome build was modified at the ura4 locus to reflect the ura4-UTRDIS/kanMX (1491A) or ura4-UTRD/kanMX (1491C) genotypes. Fasta files for each of these genomes are included.
The modified genome build 'PombeGenome_1491A.fasta' was used to generate the 1647A_terDISPkan*.igv files
The modified genome build 'PombeGenome_1491C.fasta' was used to generate the following files;
1647C_terKOkan*.igv,
1647C_clr4D_terKOkan*.igv ,
1647C_cid14D_terKOkan*.igv,
1647C_rdp1D_terKOkan*.igv files
The rest of the igv files were generated using wild type genome build.
Genome_build: S. pombe h- (GCA_000002945.1)
Supplementary_files_format_and_content: IGV-viewable files are tab delimited and include reads per million at each nucleotide position.
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| Submission date |
Nov 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ruby Yu |
| Organization name |
Harvard University
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| Department |
Cell Biology
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| Lab |
Danesh Moazed
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| Street address |
240 Longwood Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL15167 |
| Series (2) |
| GSE52534 |
Role of 3' UTR in spreading of siRNAs |
| GSE52535 |
Determinants of heterochromatic siRNA biogenesis and function |
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| Relations |
| BioSample |
SAMN02413283 |
| SRA |
SRX379598 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1269377_1491C_dcr1D.siRNA.3.norm_a.igv.gz |
2.5 Mb |
(ftp)(http) |
IGV |
| GSM1269377_1491C_dcr1D.siRNA.3.norm_s.igv.gz |
2.4 Mb |
(ftp)(http) |
IGV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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