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Sample GSM7021651

Query DataSets for GSM7021651

Status

Public on Feb 01, 2024

Title

Sham

Sample type

SRA

Source name

Walker256

Organism

Rattus norvegicus

Characteristics

tissue: spinal cord cells
injected cell type: hydrostatic cancer cells
injected cell line: Walker256
treatment: sham

Growth protocol

Walker256 cells in the logarithmic phase were collected and washed with PBS 3 times. The cell concentration was adjusted to 1×107cells/ml and 0.5ml was injected into the peritoneal cavity of SD rats. 9-12 days later, the ascites was centrifuged to collect the Walker 256 cells and 5μl (4×105 cells) of cell suspension was injected into the bone marrow cavity 0.5cm below the tibia, while the sham operation group was injected with Walker 256 cells boiled for 20 minutes and underwent the same surgical procedure as above. The BCP group and the sham control group were each composed of 3 animals, which were kept for 21 days after surgery and subsequently anaesthetized and sacrificed.

Extracted molecule

total RNA

Extraction protocol

Total RNA from the cells was isolated using the Trizol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions, followed by a DNase I digestion for 30 minutes to remove genomic DNA
Total RNA was treated with the RiboMinus kit (Life Technologies) to remove the ribosomal RNAs; RNAs were incubation with 16 units RNase R (Sigma–Aldrich, St. Louis, MO, USA) for 15 min at 37℃ to deplete linear and enrich circRNAs; The remaining RNAs were fragmented and reverse-transcribed using random primers and reverse transcriptase; Second strand cDNA synthesis was completed using DNA polymerase I and RNase H. Sixth, the Illumina series KAPA Library Preparation Kit (Kapa Biosystems) was used to perform end repair, dA-tailing, and adapter ligation; The dUTP-marked second strand was digested at 37 °C for 30 min using Uracil Specific Excision Reagent (USER) enzyme (New England Biolabs); The cDNA was enriched by PCR amplification to create the final cDNA libraries.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

circRNA-seq

Data processing

Illumina CASAVA 1.8 software used for basecalling.
Low quality which have more than 20% low base quality bases or adapter sequence contamination reads were discarded. Then high quality reads were aligned to the reference genome using BWA mem alignment module. CircRNA identify and characterize was used the CIRI2 software based on the following process. First, reads aligning contiguously to the genome without any trimming were discarded. The remaining reads were used for circRNA candidate detection. Next, terminal sequences (anchors) were extracted and re-aligned independently to find unique anchor positions. Anchors that aligned in the reverse orientation (head-to-tail) indicated circRNA splicing (back-splicing).
The following criteria were used to filter the resulting splicing events: 1) GT/AG signal flanking the splice sites; 2) unambiguous breakpoint detection; 3) no more than two mismatches in the extension procedure; 4) the breakpoint reside a maximum of two nucleotides inside an anchor; 5) less than two independent reads support the head-to-tail splice junction.
Counts of reads mapping across an identified back-splice are normalized by number of reads mapping (spliced tags reads per million mapping, TPM), which allows expression comparisons between two circRNAs. Differential expression analysis of two samples was performed using the previous statistics model. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. CircRNA with an adjusted P-value <0.05 were assigned as differentially expressed.
Assembly: rn6
Supplementary files format and content: tab-delimited text files include TPM values for each Sample

Submission date

Feb 02, 2023

Last update date

Feb 01, 2024

Contact name

chen wei fen

E-mail(s)

chenwf0927@gmail.com

Phone

13713896659

Organization name

Forevergen