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Sample GSM4006691 Query DataSets for GSM4006691
Status Public on Aug 02, 2022
Title Control Mus hippocampus 2
Sample type SRA
Source name Control Mus hippocampus
Organism Mus musculus
Characteristics strain: C57BL/6
stress: Control
tissue: brain
tissue region: hippocampus
Treatment protocol CUS used in this study followed the procedure previously validated in mice.The stressors included (1) food deprivation for 24 h, (2) water deprivation for 24 h, (3) overnight illumination, (4) absence of sawdust in cage for 24 h, (5) moistened sawdust with water for 24 h, (6) forced swimming at 8℃ for 5 min, (7) tail nipping (1 cm from the tip of the tail), (8) physical restraint for 6 h, and (9) 45° cage-tilt along the vertical axis for 3 h.Mice were exposed to various randomly scheduled, low-intensity social and environmental stressors 2-3 times a day for 4 weeks.
Growth protocol Adult male C57BL/6J mice (24.0–26.0 g, 8–10 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and randomly assigned to experimental groups. All animals were housed under a constant temperature and humidity and a 12-h light/12-h dark cycle, with the lights turned on at 7:00 AM. Food and water were available ad libitum.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Agilent 2100 with RIN number >7.0.
Poly(A) RNA is purified from total RNA(5ug) using poly-T oligo-attached magnetic beads using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA, which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP. An A-base is then added to the blunt ends of each strand, then ligated to modified Illumina multiplex barcode adapters, which including custom Unique Molecular Identifiers for minimizing sequence-dependent bias and amplification noise according to (Shiroguchi et al. 2012), and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, The ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300 bp (±50 bp). At last, we performed the paired-end sequencing on an Illumina Novaseq TM 6000 following the vendor's recommended protocol.
Library strategy ncRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina NovaSeq 6000
Description Ctrl2
Data processing Mainstream softwares (CIRCexplorer and Mapsplice) were used to identify circRNA transcripts through site recognition and analyze the splicing of circRNA forming from exon or intron. If the species of the sample exists in circBase, all identified circRNA will be compared with known circRNA, and the number and proportion of known circRNA in sequencing results will be counted.
The expression of known circRNA and new circRNA in each sample was counted and normalized by TPM.The gene expression pattern of the sample can be examined as a whole by TPM density distribution.Similarity between samples can be preliminarily inferred from the overall gene expression pattern.
According to the read count value of circRNA in each sample, the differential expression of circRNA among samples was analyzed. DESeq or DEGseq software could be used according to whether there were biological duplicates.The differences of circRNA can better reflect the reasons for the formation of biological phenotypes.
The mapped reads of each sample were assembled using StringTie. Then, all transcriptomes from Samples were merged to reconstruct a comprehensive transcriptome using perl scripts. After the final transcriptome was generated, StringTie and edgeR was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for circRNA by calculating FPKM.
Genome_build: mm9
Supplementary_files_format_and_content: FPKM values for each Sample
Submission date Aug 03, 2019
Last update date Aug 02, 2022
Contact name Rongrong Huang
Phone 15905173096
Organization name Southeast University
Street address 87 Dingjiaqiao
City Nanjing
State/province Jiangsu
ZIP/Postal code 210009
Country China
Platform ID GPL24247
Series (1)
GSE135343 circRNA sequencing facilitates quantitative analysis of CUS mice hippocampus and Control mice hippocampus
BioSample SAMN12492014

Supplementary data files not provided
Raw data are available in SRA
Processed data are available on Series record

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