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Sample GSM7872512

Query DataSets for GSM7872512

Status

Public on Aug 14, 2024

Title

E21B-2

Sample type

SRA

Source name

early-stage ovarian cancer

Organism

Gallus gallus

Characteristics

tissue: early-stage ovarian cancer

Extracted molecule

total RNA

Extraction protocol

All samples were predigested at 56◦C for 1 hour, after which the buffer was changed and then a second 12- hour digest was performed. Digests from methods B and C were centrifuged at 6000 ×G for 1 minute, after which 500 μl supernatant was mixed 1:8 with a binding buffer. DNA bound to the columns was washed with 800 μl buffer PE (Qiagen), then eluted using 2 washes in 17 μl buffer EB (Qiagen)—each with an incubation for 5 minutes at 37◦C. Prior to library construction, small aliquots of each extract were analysed on an Agilent 2200 TapeStation HS chip (Agilent Technologies, Palo Alto, CA, USA) for fragment size estimation and molar concentration.
Library blanks and index PCR blanks were also included to evaluate the potential contaminations during the library building process. Adapters were first diluted to 500 μM with ×1 TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0, SigmaAldrich). Subsequently, an equimolar concentration of each pair of long and short adapters was mixed together and hybridized through incubation at 95◦C for 1 minute, followed by a decrease in temperature with 0.1◦C/s from 95◦C to 12◦C. After hybridization, adapters AD1 and AD2 were mixed and diluted at a concentration of 10 μM prior to their use in the library construction.
First, the libraries were qPCR-quantified using the CommonprimerBGI forward primer and 1 of the indexed reverse primers. Second, subsequent index PCR amplifications used 8 to 15 cycles with CommonprimerBGI forward primer and the indexed reverse primers. Third, because several of the BGISEQ-500 libraries exhibited residual adapter dimers after the initial purification post-index PCR, each purified BGISEQ-500 library was split to 2 aliquots, and 1 of each aliquot was subject to an extra purification to remove any residual primer dimers. All amplified libraries were subsequently sent to BGI for circularization and sequencing on the BGISEQ-500 platform. For circularization, PCR products with different barcodes were pooled together at equimolar concentration to yield a final amount of 80 ng.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Data processing

The raw reads obtained from the HiSeq 2500 and BGISEQ-500 were analysed using FastQC (FastQC, RRID:SCR 014583) to compute the quality metrics of the reads, such as base sequence qualities, base sequence content, %GC, and sequence composition.
Once the read qualities were verified using FastQC (FastQC, RRID:SCR 014583), we used the PALEOMIX pipeline (PALEOMIX, RRID:SCR 015057) [18] to trim the adapter sequences, trim Ns and low-quality bases from the ends of reads, estimate ancient DNA damage, and finally map the trimmed reads to the reference genome.
AdapterRemoval (v. 2.1.3) was used to trim the adapter sequences from the ends of the reads using the default mismatch rate of 1/3.
In addition, bases with a quality score of less than 2 and unidentified bases (Ns) at the ends of reads were trimmed.
Finally, only reads that were longer than 25 bases were retained for downstream analyses.
Supplementary files format and content: The trimmed reads were mapped to the chicken reference genome using the mem algorithm in bwa (BWA, RRID:SCR 010910; v. 0.7.10), using the default settings for the mapping algorithm.
Supplementary files format and content: The mapped reads were subsequently processed using the GATK (v. 3.3.0) indel realigner (GATK, RRID:SCR 001876) to fix the alignment issues arising from the presence of short indels at the beginnings and ends of reads.

Submission date

Oct 30, 2023

Last update date

Aug 14, 2024

Contact name

guoqiang zhu

E-mail(s)

bio.guoqiang.zhu@gmail.com

Organization name

sichuan university

Street address

No. 24, Section 1, South Section 1, First Ring Road