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Sample GSM5570731

Query DataSets for GSM5570731

Status

Public on Sep 16, 2021

Title

CAFR-CM2

Sample type

SRA

Source name

Panc1 cell

Organism

Homo sapiens

Characteristics

cell line: Panc1

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from cells using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manual instructions. Cells were washed with PBS and the 1.5ml Trizol reagent was added into the 2ml tube containing the cell pellets. The mix was centrifuge at 12000×g for 5min at 4°C. The supernatant was transferred to a new 2.0ml tube which was added 0.3 ml of Chloroform/isoamyl alcohol (24:1) per 1.5ml of Trizol reagent. After the mix was centrifuged at 12000×g for 10min at 4°C, the aqueous phase was transferred to a new 1.5mLtube which was add equal volume of supernatant of isopropyl alcohol. The mix was centrifuged at12000×g for 20min at 4°C and then removed the supernatant. After washed with 1 ml 75% ethanol, the RNA pellet was air-dried in the biosafety cabinet and then dissolved by add 25µL~100µL of DEPC-treated water.
Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). According to the manufacturer’s instructions, the first step involves the removal of ribosomal RNA (rRNA) using target-specific oligos and RNase H reagents to deplete both cytoplasmic (5S rRNA,5.8S rRNA, 18S rRNA and 28S rRNA) and mitochodrial ribosomal RNA (12S rRNA and 16SrRNA) from total RNA preparations. Following SPRI beads purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. This process removes the RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. After UDG treatment, the incorporation of dUTP quenches the second strand during amplification. The products are enriched with PCR to create the final cDNA library. The libraries were assessed quality and quantity in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The qualified libraries were sequenced pair end on the BGISEQ-500/ MGISEQ-2000 System (BGI-Shenzhen, China).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MGISEQ-2000RS

Description

pre-treated with CAFR-CM

Data processing

The data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
The clean reads were mapped to the reference genome using HISAT2(v2.0.4)
Ericscript (v0.5.5) and rMATS (V3.2.5) were used to detect fusion genes and differential splicing genes (DSGs), respectively
Bowtie2 (v2.2.5) was applied to align the clean reads to the gene set, a database built by BGI (Beijing Genomic Institute in ShenZhen), in which known and novel, coding and noncoding transcripts were included,
Expression level of gene was calculated by RSEM (v1.2.12)
Differential expression analysis was performed using the DESeq2(v1.4.5) with Q value ≤ 0.05.
GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expression gene was performed by Phyper based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05) by Bonferroni
Genome_build: Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.p13)
Supplementary_files_format_and_content: Matrix table with FPKM for every gene and every sample

Submission date

Sep 09, 2021

Last update date

Sep 16, 2021

Contact name

xiang zhang

E-mail(s)

zhangx256@mail2.sysu.edu.cn

Phone

15521137982

Organization name

Sun Yat-sen Memorial Hospital