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Sample GSM1606312

Query DataSets for GSM1606312

Status

Public on Jan 07, 2016

Title

ClutchA_ribozero_23_hpf

Sample type

SRA

Source name

Whole embryo, 23hpf, RiboZero RNA

Organism

Xenopus tropicalis

Characteristics

genotype/variation: wild type
tissue: whole embryo
clutch: A
time (hpf): 23

Growth protocol

We performed IVF and flooding of the eggs with 1/9xMMR after application of sperm to eggs was considered time zero. All fertilizations and subsequent culturing of embryos were performed in a temperature-controlled room maintained at ~24oC, with fluctuations +/- ~1oC over the entire timecourse. Unfertilized eggs and embryos were dejellied and embryos were cultured at low density, ~100 embryos per 150-mm dish containing ~120-150mls of 1/9thX MMR.

Extracted molecule

total RNA

Extraction protocol

For most samples, 10 embryos/timepoint were thoroughly homogenized in 200 ml Trizol® and frozen at -80oC, with the exception of egg, 0.5, 1, 1.5, 2, 2.5 and 3hpf timepoints, which were sampled at 25, 30, 20, 20, 20, 15 and 15 embryos, respectively.
For the Clutch A timecourse, ERCC Spike In Mix 1 (10ul) (lot 1205006) was diluted in DEPC-treated water to a final volume of 1045ul. Diluted spike in mix was then pipetted directly into the Trizol homogenates such that 1ul was delivered per embryo, which in most cases required delivery of 10ul diluted spike in mix/sample, with the exception of the first several timepoints. Spike in mix was pipetted into samples in the order of their chronology. Total RNA was purified from the embryo homogenate according to the manufacturer’s recommendations. Following isopropanol precipitation, RNAs were resuspended in DEPC-treated water and any contaminating genomic DNA was removed by a subsequent overnight precipitation in 5M LiCl at 4oC. RNA was pelleted and washed twice with 70% ethanol made with DEPC-treated water. All RNAs were resuspended and quantitated by Bioanalyzer. RNA quality was assessed by Bioanalyzer and RNA Integrity Numbers were all in excess of 9.
For the Clutch B timecourse, ERCC spike in mix 1 (lot 1306008) was similarly diluted and delivered to all Trizol® homogenates, except the pipetting was done in reverse order to the chronology of the samples. Furthermore, we added an additional spike in mix that was pipetted independently and in order of sample chronology. Ambion ArrayControl™ spikes were used. Two microliters of spikes 1, 2 and 4 were diluted to a final volume of 82ul in DEPC-treated water to create a solution of 2.439ng for each ArrayControl spike-in RNA per microliter. Approximately 5.125ul of this dilution was then further diluted into DEPC-treated water to a final volume of 1000ul as verified by weight to create a solution of ~12.5pg/ul. One microliter per embryo (~12.5pg) was added to Trizol® homogenates by pipetting of 10ul per tube except in the cases of the 0-3hpf samples. In both timecourses these early timepoint spike ins were pipetted by multiple rounds of 10ul (for 20 or 30ul delivery), or readjustment to 5ul (for 15ul delivery). RNAs were subsequently isolated as described above except we omitted the LiCl precipitation step. RNAs were similar quantitated and quality evaluated.
RNA quality and concentration were evaluated by running total RNA on an Agilent RNA Nano Bioanalyzer chip. All samples had an RNA integrity number (RIN) greater than 9. Ribosomal RNA was removed via polyA-selection or ribosomal RNA depletion. For polyA-extraction, polyadenylated RNA transcripts were selected from the total RNA using two rounds of enrichment with oligo(dT)25 beads (Invitrogen). Ribosomal-depletion was completed with the Ribo-Zero (Human/Mouse/Rat) Magnetic Ribosomal rRNA Removal kit (Epicenter) according to the manufacturer's recommendations. The supernatant from both approaches was purified and concentrated using RNAclean XP beads (Beckman Coulter). Following selection, the RNA was fragmented to approximately 140 base pairs and first-strand synthesis was completed with SuperScript III reverse transcriptase (Invitrogen) using random hexamers. Magnetic AMPure XP beads (Beckman Coulter) were used to purify the cDNA and remained with the sample throughout sequencing library construction. During preparation, DNA is selectively precipitated by weight and re-bound to the beads through the addition of a 20% polyethylene glycol, 2.5 M NaCl solution. We used T4 DNA polymerase and T4 polynucleotide kinase to blunt ends and phosphorylate the fragments. The large Klenow fragment then added a single adenine residue to the 3' end of each fragment, custom adapters (IDT) are ligated using T4 DNA ligase, and, for strand-specific libraries, UDG removes uracil residues. The product is then PCR amplified using custom-made primers (IDT) containing unique 6 bp indices.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RiboZero RNA
clutch_A_ribozero/Sample_BlitzKhokhaXtRibo-ExProfile_46_006

Data processing

Read pairs were aligned to Xenopus tropicalis v7 genome along with known off-genome gene sequences and exogenouns ERCC and ArrayControl spike in sequences using tophat v2.010 and parameters "-r 13 --mate-std-dev 60 -g 200 --report-secondary-alignments".
Abundances were estimated using cufflinks v2.1.1 with parameters ""--compatible-hits-norm --max-bundle-frags 10000000 -b xt7.fa -u --max-mle-iterations 50000". This is with exception of mir427, in which reads aligning at least once to the locus and nowhere else in the genome were counted once to calculate the FPKM for the entire locus. Transcript FPKM were converted to TPM.
Relative gene abundances in TPM were absolute normalised against ERCC spiked RNA standards, resulting abundances are reported in transcripts per embryo.
Genome_build: Xtropicalis_v7
Supplementary_files_format_and_content: Tab-delimited text files containing relative and absolute normalised gene and isoform abundances.

Submission date

Feb 09, 2015

Last update date

May 15, 2019

Contact name

Nick Owens

Organization name

University of Exeter Medical School

Street address

Barrack Road

City

Exeter