|
| Status |
Public on Sep 23, 2013 |
| Title |
stage 8 small RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
Whole embryos
|
| Organism |
Xenopus tropicalis |
| Characteristics |
genotype/variation: wild-type
developmental stage: Embryonic stage 8
tissue: Whole embryos
molecule subtype: small RNAs 18-30 nucleotides
|
| Treatment protocol |
Embryos were not treated.
|
| Growth protocol |
X. tropicalis embryos were obtained by in vitro fertilization, de-jellied in 2.2% cysteine and cultured in 1/20 x MMR + gentamycin until the indicated stage as described (Khokha et al. 2002).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was obtained by Trizol extraction from X. tropicalis embryos and treated with DNAse I (Worthington Biochemical Corporation). Small RNA libraries were prepared as described (Hafner 2008). Briefly, small RNAs of 18–30 nt were size-selected by gel purification and RNA adapters ligated on using T4 RNA ligase (Promega). cDNA was synthesized and used as a template to amplify the small RNA library by PCR. The PCR library was adjusted to 10 nM concentration and single-end sequenced using the small RNA sequencing primer (Illumina) on the Illumina platform on a Genome Analyzer IIx at the Cambridge Research Institute.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
small RNA-seq
Sample 1
|
| Data processing |
small RNA-seq reads were trimmed to remove sequencing adapters and aligned to the X. tropicalis genome allowing for zero mismatches using the bwa alignment program (Li and Durbin 2009).
Normalised abundance measurements were generated to allow comparison across the libraries using a linear scaling whereby the raw reads were multiplied by the greatest number of genomic alignments in any library divided by the number of genomic alignments in the library of interest.
mRNA-seq reads were aligned to the X. tropicalis genome using bowtie (Langmead et al. 2009) allowing up to 2 mismatches. mRNA-seq reads over splice junctions were aligned using TopHat (Trapnell et al. 2009).
mRNA-seq reads over splice junctions were aligned to the X. tropicalis genome using TopHat (Trapnell et al. 2009). Up to two sequence mismatches were permitted.
Normalised abundance measurements of mRNA reads were generated using the reads per kilobase per million of exon (RPKM) method for each gene annotated in RefSeq to normalize for transcript length and total read number (Mortazavi et al. 2008).
Genome_build: X. tropicalis genome, Joint Genome Institute assembly version 4.1 (Hellsten et al. 2010)
Supplementary_files_format_and_content: BED files were generated using bedtools software and contain alignments of trimmed small RNA reads to the X. tropicalis genome or alignments of trimmed mRNA-seq reads including reads spanning exon-exon junctions.
|
| |
|
| Submission date |
Jun 08, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Caroline Hill |
| E-mail(s) |
[email protected]
|
| Phone |
+44 20 7269 2941
|
| Organization name |
Cancer Research UK London Research Institute
|
| Lab |
Developmental Signalling
|
| Street address |
44 Lincoln's Inn Fields
|
| City |
London |
| ZIP/Postal code |
WC2A 3LY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13741 |
| Series (1) |
| GSE38605 |
Genome-wide small RNA profiling and mRNA profiling of Xenopus embryos |
|
| Relations |
| SRA |
SRX152563 |
| BioSample |
SAMN01041907 |
