All in one reverse transcription mastermix

Total RNA was isolated using Trizol (Life Technologies, South San Francisco, CA USA) according to the manufacturer’s instructions. Total RNA (2 μg) was reverse transcribed using the 5× All-In-One reverse transcription MasterMix (abm, Vancouver, Canada). Quantitative real-time PCR was performed by mixing cDNA, gene-specific primers, and EvaGreen 2× qPCR MasterMix (abm, Vancouver, Canada) in the QuantStudio 3 Real-Time PCR System (Applied Biosystems). Primer sequences are provided in the S Table 1. For RNA-sequencing assays, the quality of the purified RNA was tested on Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit) (BGI, Shenzhen, China). Libraries for cluster generation and DNA sequencing were prepared following an adapted method from the BGISEQ-500 platform. The low quality reads (more than 20% of the bases qualities are lower than 10) were filtered to get the clean reads. Then those clean reads were assembled into Unigenes, followed with Unigene functional annotation, SSR detection and calculation of the Unigene expression levels and SNPs of each sample. Finally, DEGs (differential expressed genes) were identified between samples and from clustering analysis and functional annotations.

Total RNA was extracted by using Trizol (Life Technologies, South San Francisco, CA USA), and total RNA (2 μg) was reverse transcribed into cDNA by using the 5× All-In-One reverse transcription MasterMix (ABM, Vancouver, Canada). Quantitative PCR (qPCR) was performed by mixing the cDNA, gene-specific primers, and EvaGreen 2× qPCR MasterMix (ABM) in the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences were:

GHET1-F: CGCAAGGTACCAGAGAGCCG;

GHET1-R: AGTGGCGCTTGo CTGGGATTT;

ITGAM-F: GCTTTGGTGGCTTCCTTGTG;

ITGAM-R: TAGTCGCACTGGTAGAGGCT;

miR-105-F: GTGCATCGTGGTCAAATGCT;

miR-105-R: ACACCGTAGCACATGCTCAA

RAP2B-F: AAGCCTCGGTAGACGAGCTA;

RAP2B-R: GTCGGATGCGTTTGGCTTTT;

GAPDH-F: CTGGGCTACACTGAGCACC;

GAPDH-R: AAGTGGTCGTTGAGGGCAATG.

Total RNA was isolated using Trizol (Life Technologies, South San Francisco, CA USA) according to the manufacturer’s instructions. Total RNA (2 μg) was reverse transcribed using the 5× All-In-One reverse transcription MasterMix (abm, Vancouver, Canada). Quantitative real-time PCR was performed by mixing cDNA, gene-specific primers and EvaGreen 2× qPCR MasterMix (abm, Vancouver, Canada) in the QuantStudio 3 Real-Time PCR System (Applied Biosystems). Primer sequences are provided in the Supplementary Information.