|
| Status |
Public on Aug 04, 2014 |
| Title |
RNAseq_single_embryo_replicate2 |
| Sample type |
SRA |
| |
|
| Source name |
embryo
|
| Organism |
Xenopus laevis |
| Characteristics |
developmental stage: NF stage 10.5
tissue: whole embryo
|
| Growth protocol |
A single X.laevis female was injected with 700u of human choriogonadotropin (Brevactid 1500 I.E., Ferring) and eggs were fertilized in vitro. Turned, uncleaved eggs were collected 60 minutes post fertilization, gastrulating embryos were staged according to Nieuwkoop and Faber and collected at stage 10.5. Embryos were dejellied in 2% cysteine hydrochloride (Sigma) pH8 in 0.25X MMR. Each egg/embryo was collected in an independent tube, snap-frozen in liquid nitrogen and stored at -80°C until RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRizol® Reagent (Ambion) and RNeasy Kit (Quiagen), and rRNA depleted with Ribo-Zero™ rRNA Removal Kits (Epicentre). rRNA-depleted RNA was then heat-fragmented in 40mMTris-acetate, 100mM potassium acetate and 30mM magnesium acetate (pH8.2) and used for cDNA synthesis. First cDNA strand was obtained from random examers primers using Superscript III (Invitrogen); second strand was made with E.coli DNA polymerase I (NEB), E.coli DNA ligase (NEB) and T4 DNA polymerase (Promega). Purified cDNA was used for strand-specific Illumina sample preparation.
Libraries were constructed according to standard Illumina protocols. Briefly cDNA was ligated with adapter sequences, size selected (300bp), and amplified by PCR. For the embryo samples purified cDNA was used for strand-specific Illumina sample preparation using Kapa Hyper Prep Kit, according to manufacturer protocol. To confer strand-specificity a USER enzyme (NEB) digestion step was performed prior to library amplification.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina BCL files were converted to FASTQ and demultiplexed using Illumina CASAVA 1.8.2.
Sequences were mapped to JGI 7.1 using gmap version 2012-04-21 with parameters -m 1 -B 4 -t 8 -N 1 -E 100 -w 100000 -n 1.
Transcript abundance and differential expression was calculated by quantifying the Reads Per Kilobase of transcript per Million reads mapped (RPKM value) using Cufflinks (version 2.1.1).
To compare the transcriptomes of the X. laevis eggs and embryos 10.5 the RNA-seq data were concatenated for each developmental stage using the BamTools API, followed by the removal of random sequences with SAMtools v0.1.18 until the amount of mapped reads were similar. These files were analyze using Cufflinks version v2.1.1; results are in isoforms.fpkm_tracking.reduced.txt.
Genome_build: Xenopus leavis JGI 7.1 obtained from Xenbase (http://www.xenbase.org).
Supplementary_files_format_and_content: Isoforms.fpkm_tracking.individual.txt contains the Cuffdiff output for the egg and embryo replicates seperately. Isoforms.fpkm_tracking.reduced.txt contains the Cuffdiff output for the total egg and embryo reads.
|
| |
|
| Submission date |
Jul 08, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Gert Jan Veenstra |
| E-mail(s) |
g.veenstra@science.ru.nl
|
| Phone |
+31 24 3610541
|
| Organization name |
Radboud University
|
| Department |
Molecular Developmental Biology
|
| Street address |
Geert Grooteplein 28
|
| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL17682 |
| Series (1) |
| GSE56586 |
Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs |
|
| Relations |
| BioSample |
SAMN02905141 |
| SRA |
SRX648248 |
