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Sample GSM7157370

Query DataSets for GSM7157370

Status

Public on Apr 11, 2023

Title

Control of DEF cells-2

Sample type

SRA

Source name

Duck embryo fibroblasts

Organism

Anas platyrhynchos

Characteristics

developmental stage: embryo
cell type: fibroblasts
genotype: WT
infection: Non
time: 9 h

Extracted molecule

total RNA

Extraction protocol

1) Wash worms from a 60 mm plate with 1 ml DEPC-treated water.2) Spin down worms at 4000xg in microfuge for 1 minute.3) Remove excess water and add 1 ml Trizol reagent. Vortex and invert tube. Let sit at room termerature for 10 minutes (should see stringy-like material).4) Spin tubes in microfuge at 14K for 10 minutes at 4° C.5) Remove supernatant into fresh RNAse free eppendorf tube and add 200 µl of chloroform.6) Vortex for 15 seconds and let sit at room temperature for 3 minutes.7) Spin in microfuge at 12K for 15 minutes at 4° C.8) Carefully remove the top layer (clear) into a new RNAse free eppendorf tube and add 500 µl of Isopropanol. Invert to mix. Let sit for 10 minutes at room temperature.9) Spin in microfuge at 12K for 10 minutes at 4° C. (RNA will be seen as a nice white pellet).10) Wash pellet with 100 µl of 75% ethanol (made by diluting into DEPC-treated water).11) Spin in microfuge at 7500 xg for 5 minutes at 4° C.12) Remove supernatant and air dry pellet for 10 minutes.13) Dissolve pellet in 25 µl of DEPC-treated water. Heat for 10 minutes at 60° C to help dissolve RNA.
After total RNA was extracted by Trizol reagent kit (Invitrogen, Carlsbad, CA,USA), the RNA molecules in a size range of 18–30nt were enriched by polyacrylamide gel electrophoresis(PAGE). Then the 3’ adapters were added and the 36-44nt RNAs were enriched. The 5’ adapters were then ligated to the RNAs as well. The ligation products were reverse transcribed by PCR amplification and the 140-160bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeqTM 2500

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

DN-2

Data processing

Reads obtained from the sequencing machines included dirty reads containing adapters or low quality bases which would affect the following assembly and analysis.
Alignment and Identification of small RNA in GeneBank and Rfam database
Alignment with Genome (exon, intron, repeat sequences)
Identification of microRNA (miRNA) using miRbase database and software Mireap_v0.2
Small RNA annotation summary and miRNA expression profiles
Assembly: ncbi_GCF_003850225.1
Supplementary files format and content: all_mirna.exp_profile.txt

Submission date

Apr 08, 2023

Last update date

May 10, 2023

Contact name

Lei Fan

E-mail(s)

fanlei950412@126.com

Phone

18227588709

Organization name

South China Agricultural University