The total RNA from each sample group was extracted by using a TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA was further purified using an RNeasy mini kit (QIAGEN, Germantown, MD, USA). The degradation and contamination of total RNA were tested before the RNA library was prepared with 1% agarose gels. The RNA integrity was evaluated using a Qubit 2.0 Fluorometer and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The qualified RNA from each sample was used to construct the sequencing library using an Illumina TruSeq Stranded RNA Kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. The purified cDNA libraries were enriched using PCR. After cluster generation, all the libraries were sequenced on an Illumina HiSeq 2500 platform with paired-end 150 bp reads (Novogene Bioinformatics Institute, Beijing, China). Together, a total of 36 cDNA libraries were sequenced in this study.
Qubit 2.0 fluorometer and 2100 bioanalyzer
Manufactured by Agilent Technologies
Sourced in United States
The Qubit 2.0 Fluorometer is a compact and sensitive fluorescence-based instrument designed for the accurate quantification of DNA, RNA, and protein samples. The 2100 Bioanalyzer is a microfluidics-based platform that provides automated electrophoretic analysis of DNA, RNA, and protein samples.
Automatically generated - may contain errors
2 protocols using qubit 2.0 fluorometer and 2100 bioanalyzer
1
RNA-seq Library Preparation and Sequencing
2
ZDHHC2 Gene Sequencing Protocol
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Genomic DNA was extracted from peripheral blood samples using the Quick Gene DNA Whole Blood Kit (Tokyo, Japan). Sequencing primers covered the UTRs and all exons of ZDHHC2 (Table S1 ). Two‐staged PCR amplifications were performed with 20 ng DNA to generate a sequence library according to the manufacturer's instructions (Shanghai DynastyGene Co. Ltd). We barcoded the libraries using the unique 8 bp index for each sample. After purification using microbeads (Shanghai DynastyGene Co. Ltd), libraries were quantified by a Qubit 2.0 Fluorometer and 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed on the Illumina Xten platform following the manufacturer's protocol.
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