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Sample GSM7159027

Query DataSets for GSM7159027

Status

Public on Apr 11, 2023

Title

circRNA DEF cells infected with NDV-3

Sample type

SRA

Source name

Duck embryo fibroblasts

Organism

Anas platyrhynchos

Characteristics

cell line: Duck embryo fibroblasts
cell type: fibroblasts
genotype: WT
treatment: NDV infected (MOI = 1)
time: 9 hpi

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 extracted, total RNAs were treated with RNase R to degrade the linear RNAs, and purified using RNeasy MinElute Cleanup Kit（Qiagen）. Next, strand-specific library was constructed using VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina following the manufacture’s instructions. Briefly, ribosome RNAs were removed to retain circRNAs. The enriched circRNAs were fragmented into short fragments by using fragmentation buffer and reverse transcripted into cDNA with random primers. Second_x0002_strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP) and buffer. Next, the cDNA fragments were purified with VAHTSTM DNA Clean Beads, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. Then UNG (Uracil-N-Glycosylase) was used to digest the second-strand cDNA. The digested products were purified with VAHTSTM DNA Clean Beads, PCR amplified, and sequenced using Illumina HiSeqTM 2500

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Filtering of Clean Reads
Alignment with Ribosome RNA (rRNA) using short reads alignment tool Bowtie2
Alignment with Reference Genome by using TopHat2
Identification of circRNA by using find_circ
Assembly: ncbi_GCF_003850225.1
Supplementary files format and content: excel format: Number of raw junction read

Submission date

Apr 10, 2023

Last update date

May 01, 2023

Contact name

Lei Fan

E-mail(s)

fanlei950412@126.com

Phone

18227588709

Organization name

South China Agricultural University