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| Status |
Public on Oct 01, 2012 |
| Title |
WT_3h_nonOx |
| Sample type |
SRA |
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| Source name |
mating wild-type cells at 3h post-mixing
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| Organism |
Tetrahymena thermophila |
| Characteristics |
sample type: Whole RNAs
genotype/variation: wild type
strains: B2086 and CU428
post-mixing time: 3h
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| Growth protocol |
Wild-type Tetrahymena thermophila strains B2086 and CU428, and the complete (germ plus soma) TWI1 KO (Mochizuki and Gorovsky 2004) strains 20-1 and 20-4 were used for total RNA preparation. Mating of cells was induced as described (Gorovsky et al. 1975), and asynchronous mating initiation was limited by adding quarter of the total volume of 4x SPP at ~3 hr post-mixing.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from 2.5 x 106 mating cells at the indicated times post-mixing using the mirVana miRNA isolation kit (Ambion). For periodate oxidation of small RNAs (Akbergenov et al. 2006), the RNA pellet was dried and resuspended in 17.5 µl borax buffer (4.375 mM borax, 50 mM boric acid; pH 8.6). Next, 2.5 µl 0.2 M sodium periodate was added, and the reaction was incubated for 10 min at RT in the dark. Then, 2 µl of glycerol was added, the reaction was incubated for another 10 min at RT in the dark, and the small RNAs were purified by ethanol precipitation. The cDNA library of small RNAs was constructed as described (Hafner et al. 2008) with some modifications. In brief, approximately one-tenth of the purified RNA (with or without periodate oxidation) was mixed with radiolabeled 26- and 32-nt oligoribonucleotides (5’-GUC GUA CGC GGA AUA GUU UAA ACU GU-3’ and 5'-AUC UUG GUC GUA CGC GGA AUA GUU UAA ACU GU-3’, respectively; the PmeI restriction site is underlined), fractionated in a sequencing gel, and the gel region containing the two radiolabeled markers and a few bases on each side were excised. RNA was recovered from the gel and ligated to a 5'-adenylated 3' adapter oligonucleotide (5’-App-TCG TAT GCC GTC TTC TGC TTG-L-3’; L = 3'OH blocking group; a gift from Markus Landthaler from the Tuschl lab) using truncated T4 Rnl2tr (NEB). After gel fractionation and purification as above, the product was ligated to the 5' adapter oligoribonucleotide (5'-GUU CAG AGU UCU ACA GUC CGA CGA UC-3') using T4 RNA ligase (Ambion). The gel-purified final ligation product was then reverse-transcribed using the primer 5’-CAA GCA GAA GAC GGC ATA CGA-3’ and PCR-amplified using that reverse transcription primer and the primer 5’-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3’. After phenol-chloroform extraction followed by ethanol precipitation, the PCR product was digested with PmeI (to cut the size markers) and fractionated by agarose gel electrophoresis, and the uncut PCR product was purified from the gel. The amplified cDNA libraries were mixed with phiX174 phage DNA at one-tenth of the amount of DNA, and single-read 36-base sequences were generated on the Illumina GAII platform obtaining 2-30 million reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
WT_3h_nonOx
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| Data processing |
Following removal of the adapter sequences from the sequence reads, the reads from rRNAs, tRNA, snRNAs, the mitochondrial genome, and mixed phiX174 phage DNA were removed. To study the base composition and thermodynamic stability of small RNAs, reads containing any bases other than A, C, G or T were excluded and the remaining reads were collapsed to non-redundant datasets. To study thermodynamic stability, 29 nt RNA sequences were mapped to the LMR genomic region (corresponding to bases 64,932-165,807 of the supercontig 231 in the 2nd assembly of the Tetrahymena thermophila micronuclear genome, obtained from Broad Institute Web site: http://www.broadinstitute.org/annotation/genome/Tetrahymena.1/MultiHome.html) without allowing any mismatches. Then, the passenger strand scnRNA sequences were estimated. The local thermodynamic stabilities (kcal/mol) of 4 base pairs at the ends of the guide and passenger duplexes were calculated using the free energy parameters at 37ºC in 1 M NaCl (Freier et al. 1986) without including the initiation value. To study potential trimming and tailing reactions on small RNAs, 24-32 nt RNA sequences were mapped to the LMR genomic region by allowing two mismatches, and the mismatched positions in the scnRNAs were analyzed.
The processed data file linked below represents base composition of 29 nt small RNAs from wild-type cells at 3 h post-mixing, prepared with no oxidation process
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| Submission date |
May 01, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kazufumi Mochizuki |
| E-mail(s) |
kazufumi.mochizuki@igh.cnrs.fr
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| Organization name |
Institute of Human Genetics, CNRS
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| Street address |
141 rue de la Cardonille
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| City |
Montpellier |
| ZIP/Postal code |
34090 |
| Country |
France |
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| Platform ID |
GPL15515 |
| Series (1) |
| GSE37696 |
Analysis of conjugation-specific 26-32nt siRNAs of Tetrahymena thermophila |
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| Relations |
| SRA |
SRX145841 |
| BioSample |
SAMN00990800 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM925729_WT_3h_nonOx_29nt.txt.gz |
1.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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