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Sample GSM5703571

Query DataSets for GSM5703571

Status

Public on Jul 26, 2022

Title

4 [406 2019]

Sample type

SRA

Source name

SVI oocyte

Organism

Xenopus laevis

Characteristics

cell type: Oocyte
treatment: not-injected
rip antibody: Anti-HA (3F10) High Affinity (ROAHAHA)
sequencing_batch: 201903

Treatment protocol

140 – 120 fully-grown oocytes were microinjected with 46nL of 50 ng/mL in vitro transcribed polyadenylated RNAs per condition.

Growth protocol

Defolliculated isolated oocytes were kept in Modified Bath Saline media (MBS: 88 mM NaCl, 1 mM KCl, 1 mM MgSO4, 2.5 mM NaHCO3, 0.7 mM CaCl2, pH 7.8) at 18ºC.

Extracted molecule

total RNA

Extraction protocol

16-hours post microinjection, live oocytes were lysed by pipetting in 9 μL/oocyte cold IP lysis buffer (20 mM Tris-HCl pH 8, 100 mM NaCl, 0.4% NP40, 1 mM EDTA, 1 mM MgCl2, 1x Complete EDTA-free protease inhibitors (Roche)). Homogenates were clarified by centrifugation for 10 minutes at 12000 g 4ºC. 1 μL of 10x H1K phosphatase inhibitors (80 mM sodium β-glycerophosphate, 0.5 mM sodium orthovanadate) was added per 9 μL of recovered aqueous phase and a second clarification step was performed. 50 μL of clarified lysate were put aside for protein input, 50 μL for RNA input and 600-800 μL were used for immunoprecipitation. HA-conjugated magnetic beads (Pierce Anti-HA Magnetic Beads, 88837, Thermo Fisher Scientific) were washed twice in PBS and twice in lysis buffer prior to use. A proportion of 150 μL bead-slurry per 1 mL clarified lysate was used. The clarified lysates were supplemented with 0.5 U/μL of Ribolock (Thermo Fisher Scientific) and incubated with the HA-beads for 2 hours on a wheel at 4ºC. The beads were washed thrice with one volume of Ribolock-supplemented cold lysis buffer. 1/10 of the lysate-bead-slurry was devoted to protein extraction and 9/10 to RNA extraction. Following the third wash, the RNA was eluted from the beads by Proteinase K digestion. Briefly, the beads slurry or inputs, both in 50 μL of Ribolock-supplemented IP lysis buffer, were incubated in 400 μL of Proteinase K buffer (200 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% SDS, 40 U/μL Ribolock) with 200 μg/mL Proteinase K (3115887001, Proteinase K, recombinant, PCR grade, Roche) for 30 minutes at 37ºC. Subsequently, the RNA in the supernatant was purified by organic phase extraction using Trizol Reagent (Thermo Fisher Scientific) and chloroform followed by ethanol precipitation. The pellet resulting from precipitation was reconstituted in 40 μL (inputs) or 15 μL (eluates) of TURBO DNase premix (1x TURBO buffer, AM1907, Invitrogen, supplemented with 1 U/μL Ribolock) and treated with 1 μL TURBO DNase for 30 minutes at 37ºC. The enzyme was inactivated following manufacturer’s instructions.
mRNA-purification from 0.75 and 1.7 µg of RNA was applied to input samples using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). No mRNA purification was performed on the IP samples. NGS libraries for RNA-seq were prepared using the kit NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) and 8-10 cycles of library amplification.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

Replicate#04, not-injected
0450_32672_CTTGTA

Data processing

FastQ files were aligned against the Xenopus laevis genome (UCSC version 9.2, excluding chrUn chromosomes), with Bowtie2 2.2.2, 1 mismatch and reporting best alignment site per read.
Gene regions were extracted from the Xenbase Xenopus laevis 9.2 annotation GFF, using the makeTxDbFromGFF function from the GenomicFeatures 1.34.8 and R 3.5.1.
Gene level counts were generated with the featureCounts function from the Rsubread package 1.32.4 in R3.5.1, using options allowMultiOverlap=TRUE, ignoreDup=FALSE, countMultimappingReads=FALSE,minMQS=1.
Genome_build: Xenopus laevis genome (UCSC version 9.2, excluding chrUn chromosomes)
Supplementary_files_format_and_content: Excel XLSX file with per-sample Xenbase Xenopus laevis 9.2 gene-level counts.

Submission date

Nov 25, 2021

Last update date

Jul 26, 2022

Contact name

Oscar Reina Garcia

E-mail(s)

[email protected]

Organization name

IRB Barcelona

Department

Biostatistics and Bioinformatics