 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 05, 2013 |
| Title |
scnRNAs co-immunoprecipitated with HA-Twi1p |
| Sample type |
SRA |
| |
|
| Source name |
scnRNAs co-immunoprecipitated with HA-Twi1p
|
| Organism |
Tetrahymena thermophila |
| Characteristics |
strain: HA-Twi1p-5 x HA-Twi1p-65
genotype: TWI1KO
ip antibody: anti-HA antibody (16B12, Covance)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Wild-type Tetrahymena thermophila strains B2086 and CU428 and the complete TWI1 KO strains (Mochizuki and Gorovsky 2004) were used for total RNA preparation. Twi1p was immunoprecipitated (Noto et al. 2010) from either wild-type cells using an anti-Twi1p antibody (Noto et al. 2010) or from the strains expressing HA-Twi1p and HA-Twi1p-hAGO2Lmut (Mochizuki and Kurth in press) using an anti-HA antibody (16B12, Covance). The cDNA libraries were constructed as described (Schoeberl et al. 2012). For multiplex sequencing, oligonucleotides listed below were used. Single-read 36- or 50-base sequences were generated using the Illumina GAII or HiSeq2000, respectively. Sequence reads were filtered as described (Schoeberl et al. 2012).
The 3' adapter for multiplex sequencing: 5’-App-NNN NAG ATC GGA AGA GCA CAC GTC T-3ddC; The 5' adapter for multiplex sequencing: 5'-ACA CUC UUU CCC UAC ACG ACG CUC UUC CGA UCU NNN-3'; The reverse-transcription primer for multiplex sequencing: 5’-CAA GCA GAA GAC GGC ATA CGA GAT BBB BBB GTG ACT GGA GTT CAGACG TGT GCT CTT CCG ATC T-3’ (“BBB BBB" = 6 base barcodes for maltiplex sequencing: 5'-CGT GAT-3', 5'-ACT TCG-3', 5'-GCC TAA-3', 5'- TGG TCA-3', 5'- CAC TGT-3', 5'-ATT GGC-3', 5'- GAT CTG-3', 5'- TCA AGT-3', 5'-CTG ATC -3', or 5'-AAG CTA-3'); The PCR amplification primer for multiplex sequencing: 5’-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GAT CT-3’
The RNA was gel-fractionated, and a small RNA population, including scnRNAs (26–32 nt), was used to construct the cDNA library for deep sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
HA-Twi1p_IP
|
| Data processing |
Sequence reads were filtered as described (Schoeberl et al. 2012).
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.
Genome_build: 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)
Supplementary_files_format_and_content: The .txt files represent abundance counts of the scnRNAs.
|
| |
|
| Submission date |
Jun 05, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Kazufumi Mochizuki |
| E-mail(s) |
kazufumi.mochizuki@igh.cnrs.fr
|
| Organization name |
Institute of Human Genetics, CNRS
|
| Street address |
141 rue de la Cardonille
|
| City |
Montpellier |
| ZIP/Postal code |
34090 |
| Country |
France |
| |
|
| Platform ID |
GPL15706 |
| Series (1) |
|
| Relations |
| BioSample |
SAMN02191448 |
| SRA |
SRX294297 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1154019_HA-Twi1p_IP_BaseComp.txt.gz |
8.8 Kb |
(ftp)(http) |
TXT |
| GSM1154019_HA-Twi1p_IP_length.txt.gz |
246 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
