GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5448157

Query DataSets for GSM5448157

Status

Public on Jul 14, 2021

Title

KO1EV_2

Sample type

SRA

Source name

OPM2 human myeloma cell line

Organism

Homo sapiens

Characteristics

crispr editing: FOXM1 knockout via CRISPR-Cas9
overexpressing: transfected with empty vector

Growth protocol

Cells were growing in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin

Extracted molecule

total RNA

Extraction protocol

Total RNA from OPM2 cells was extracted via RNeasy Plus Mini Kit (QIAGEN). RNA concentration, RNI value, 28S/18S and the fragment length distribution were detected via Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit).
The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T ooligoattached magnetic beads. Following purification, theThe first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligoattached magnetic beads. Following purification, the mRNA 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. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the DNBseq platform for the following data analysis study. For this step, the DNBseq platform combines the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

DNBSEQ-G400

Description

OPM2 geneexpression

Data processing

Firstly, we removed the reads mapped to rRNAs and get rawdata, then we filter the low quality reads (More than 20% of the bases qualities are lower than 10), reads with adaptors and reads with unknown bases (N bases more than 5%) to get the clean reads.
Then we map those clean reads onto reference genome(hg19), followed with novel gene prediction, SNP & INDEL calling and gene splicing detection.
Finally, we identify DEGs (differentially expressed genes) between samples and do clustering analysis and functional annotations.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited excel files include readcount values for each sample

Submission date

Jul 13, 2021

Last update date

Jul 14, 2021

Contact name

Yan Cheng

E-mail(s)

chengyan_cpu@hotmail.com

Organization name

University of Arkansas for Medical Sciences

Department

Internal Medicine