TLM2 (CRX1988310; CRA030210)
(1) Use fragmented mRNA as a template and random oligonucleotides as primers to synthesize the first strand of cDNA in the M-MuLV reverse transcriptase system; (2) Degrade the RNA strand with RNase H and synthesize the second strand of cDNA in the DNA polymerase I system using dNTPs as substrates; (3) Perform end repair on the double-stranded cDNA, add an A tail to ligate the sequencing adapter, and use VAHTS DNA Clean Beads to select cDNA fragments of approximately 250-300 bp; (4) After PCR amplification, purify using VAHTS DNA Clean Beads. (5) Library quality control: First, quantify using the Qubit 3.0 Fluorometer, then analyze using the Agilent 2100 bioanalyzer. After confirming that the insert size meets expectations, perform qRT-PCR for accurate quantification of the effective concentration of the library (the effective concentration of the library should be greater than 2 nM). (6) The library preparation kit is the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina(r) (#NR605). (7) Sequencer: Illumina XPlus.