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Sample GSM5462335

Query DataSets for GSM5462335

Status

Public on Jul 22, 2021

Title

YO_A3_input

Sample type

SRA

Source name

Yak ovary

Organism

Bos grunniens

Characteristics

developmental stage: anestrus ovary
tissue: ovary

Treatment protocol

The ovarian tissue was immediately stored in liquid nitrogen for RNA isolation. All the ovarian samples were collected from the slaughtered yak within 1 h.

Growth protocol

The ovarian tissues were collected from three healthy yaks of the same age during three different periods of the year. The samples were separately collected during the anestrus in April (A1, A2, A3), the follicular (also as estrus) phase in August (F1, F2, F3), and the pregnancy period in December (P1, P2, P3).

Extracted molecule

total RNA

Extraction protocol

The total RNA was isolated and purified using the TRIzol reagent . The amount and purity of the RNA samples were quantified using the NanoDrop ND-1000.
The Poly (A) RNA was purified from 50 μg total RNA using Dynabeads Oligo (dT) 25-61005through two rounds of purification. Then the captured RNA was fragmented into small pieces using the Magnesium RNA Fragmentation Module for 7 min at 86°C. The fragmented RNA was premixed with the Dynabeads Antibody Coupling Kit and m6A antibody（No.202003, Synaptic Systems, Germany） in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630) and incubated for 2 h at 4°C. The IP RNA was reverse-transcribed to synthesize the cDNA using the SuperScript™ II Reverse Transcriptase. Then the two-strand synthesis was performed with RNase H using the E. coli DNA polymerase I to synthesize U-labeled second-stranded DNAs. At the same time, the dUTP Solution was incorporated into the two strands, and the ends of the double-stranded DNA were filled with blunt ends. An A-base was added at each end of the fragment to connect it to the T-base overhang, and the size of the fragment was screened and purified using the AMPureXP beads. The U-labeled second-stranded DNAs were digested using a heat-labile UDG enzyme , then amplified by PCR under the following conditions: Pre-denaturation at 95°C for 3 min, denaturation at 98°C (8 cycles) for 15 s, annealing at 60°C for 15 s, extending at 72°C for 30 s, and finally extending at 72°C for 5 min to form a cDNA library of 300 ± 50 BP. At last, the 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

m6A RIP-seq

Data processing

raw data quality control performed using fastq 0.19.4.
HISAT2 2.0.4 software was used to compare the reference genome of valid data after preprocessing.
Using the peak calling software, R package exomepeak 1.9.1 performed peak scanning in the whole gene range.
Annotate these peaks using ChIPseeker 1.18.0.
Genome_build: Bos grunniens v101
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample …

Submission date

Jul 19, 2021

Last update date

Jul 22, 2021

Contact name

Xingdong Wang

E-mail(s)

wxd17339929758@126.com

Phone

17339929758

Organization name

CAAS: Chinese Academy of Agricultural Sciences