Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, United States). RNA degradation was detected using 1% agarose gel electrophoresis, while RNA purity and concentration were determined using NanoPhotometer® spectrophotometer (Implen, München, Germany). mRNA was then captured and purified from total RNA with mRNA Capture Beads (Vazyme Biotech, Nanjing, China). After fragmenting the mRNA into 100∼200 nt in the Fragmentation Buffer, mRNA was reverse-transcribed into cDNA. Synthesized cDNA was purified using DNA Clean Beads (Vazyme Biotech).
We used a sequencing strategy with optimized single-molecule barcodes to minimize sequence dependent bias and amplification noise (Shiroguchi et al., 2012 (link); Ogawa et al., 2017 (link)). The Purified cDNA was ligated to the adaptor with UMI (Unique molecular identifier) and purified using DNA Clean Beads. Finally, cDNA libraries were constructed from cDNA using PCR amplification (NEBNext Ultra RNA Library Prep Kit, Ipswich, MA, United States). The qualities of cDNA libraries were detected by the Agilent Bioanalyzer 2100 (DNA Technologies Core, Davis, CA, United States).
We used a sequencing strategy with optimized single-molecule barcodes to minimize sequence dependent bias and amplification noise (Shiroguchi et al., 2012 (link); Ogawa et al., 2017 (link)). The Purified cDNA was ligated to the adaptor with UMI (Unique molecular identifier) and purified using DNA Clean Beads. Finally, cDNA libraries were constructed from cDNA using PCR amplification (NEBNext Ultra RNA Library Prep Kit, Ipswich, MA, United States). The qualities of cDNA libraries were detected by the Agilent Bioanalyzer 2100 (DNA Technologies Core, Davis, CA, United States).
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