TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

BHK-VECs and BHK-21 cells were collected, and RNA was extracted with TRIzol reagent (Takara, China). The integrity and purity of RNA were tested using 1.5% agarose gels, NANO DROP2000 (Thermo), and Agilent 2100. Each sample was subjected to three independent replicates. Subsequent sequencing was performed at Beijing Novogene Illumina Biological Information Technology Co. Ltd. Briefly, mRNA was enriched by Oligo-attached magnetic beads (NEBNextUltraTM RNA library Prep Kit) and then fragmented into 200–400 bp. First-strand cDNA was synthesized using a random hexamer primer and M-MuLV reverse transcriptase with RNase inhibitor. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I with RNase H. After adenylation of 3′ ends of DNA fragments, NEBNext adaptors with hairpin loop structures were ligated to prepare for sequencing. An AMPure XP system (Beckman Coulter, Beverly, USA) was used to select 200–400 bp cDNA fragments. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Lastly, PCR products were purified (AMPure XP System), and library quality was assessed using the Agilent Bioanalyzer 2100 system. After library inspection qualification, HiSeq sequencing was implemented in the different libraries.

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Manufacturer protocol

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