|
| Status |
Public on Feb 21, 2018 |
| Title |
tt2MO_st26_frogF |
| Sample type |
SRA |
| |
|
| Source name |
embryo
|
| Organism |
Xenopus tropicalis |
| Characteristics |
strain: wild-type (out-bred Nigerian)
developmental stage: mid-tailbud (stage 26)
treatment: morpholino
|
| Treatment protocol |
Embryos were injected as indicated at 1-cell stage with 18 ng morpholino antisense oligonucleotide.
|
| Growth protocol |
Embryos were grown to the indicated developmental stage in 5% MMR at 25ºC.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single embryos were homogenised in 200 µl TRIzol (Thermo Fisher Scientific) by vortexing. For phase separation, 40 µl of chloroform was added to the homogenate, shaken vigorously for 15 secs before spinning for 5 mins at 16,000 g at 4ºC. The aqueous phase containing total RNA was snap-frozen in liquid nitrogen and stored at -80ºC while the interphase-organic layer was processed to extract genomic DNA for genotyping. The aqueous phases from 5 to 10 sibling embryos of shared genotype or treatment were then combined. Total RNA was precipitated with one volume of 100% ethanol and cleaned using the RNA Clean and Concentrator 5 or 25 (Zymo Research) with in-column 3 U Turbo DNase (Thermo Fisher Scientific) treatment according to the manufacturer’s instructions.
Libraries were made from ~1 µg total RNA by following version 2 of the TruSeq RNA low sample protocol (Illumina) with the following modifications. After second strand synthesis 1 µl of the remaining eluate was used to measure the cDNA concentration using the Qubit fluorometer and Qubit dsDNA high sensitivity reagents (Thermo Fisher Scientific). The resultant cDNA yield was used to estimate the number of requisite PCR cycles to generate high complexity libraries without chimera fragments. We routinely used ten PCR cycles for 10 ng cDNA and adjusted the number of PCR cycles accordingly.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
injected 18 ng t/t2 donor splice- and translation-blocking morpholino (t/t2 MO) into 1-cell stage embryos from frog F
|
| Data processing |
Bases were called using Casava version 1.8.2
Paired-end reads were aligned to a revised version of X. tropicalis gene models 7.2 and known off-genome EST assemblies including ribosomal and mitochondrial RNA by running Bowtie2 (version 2.1.0) with the following constraints: -k 200 -X 800 --rdg 6,5 --rfg 6,5 --score-main L,-.6,-.4 --no-discordant --no-mixed.
Only read pairs that uniquely align to one gene were counted. Differential expression analysis was performed with raw fragment counts excluding those belonging to ribosomal and mitochondrial RNA using DESeq2 (Love et al., 2014)
For genome browser visualisation, paired-end reads were mapped to the X. tropicalis genome assembly 7.1 and known off-genome EST assemblies using Tophat (version 2.0.10) with the following parameters: -r 77 --mate-std-dev 110 -G v7.2 (gene models of version 7.2 as used above) -g 200 --report-secondary-alignments. genome_v71.gff3.gz (genome annotation file in GFF3 format) and GO_terms_v71.txt.gz (GO term annotation file) are linked on the Series record.
Genome_build: X. tropicalis v7.1
|
| |
|
| Submission date |
Mar 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
George E. Gentsch |
| E-mail(s) |
george.gentsch@crick.ac.uk
|
| Organization name |
The Francis Crick Institute
|
| Department |
Developmental Biology Laboratory
|
| Lab |
James C. Smith
|
| Street address |
1 Midland Road
|
| City |
London |
| State/province |
Greater London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL23182 |
| Series (1) |
| GSE96655 |
Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus |
|
| Relations |
| BioSample |
SAMN06604990 |
| SRA |
SRX2642680 |
