RNA-Seq of Phaseolus vulgaris: seeds at 10 DAA- biological replicate 1
Twelve RNA-Seq libraries were constructed from three biological replicates from samples harvested at 6, 10, 14 and 18 days after anthesis (DAA). Libraries construction and sequencing was performed by NOVOGENE (Cambridge, UK). Pair-ended cDNA libraries were sequenced on Illumina NovaSeq 6000 platform following manufacturers recommendations and 150 bp paired-end reads were generated. mRNA was purified from total RNA using poly-T oligo attached magnetic beads and fragmented randomly in fragmentation buffer [NEBNext First Strand Synthesis Reaction Buffer (5X)], followed by cDNA synthesis using random hexamers and reverse transcriptase (RNase H-). Secondstrand cDNA synthesis was subsequently performed using buffer (Illumina) with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library is ready after a round of purification, adenylation of 3 ends of DNA fragments, A-tailing, ligation of sequencing adapters, size selection of cDNA fragments of preferentially 150 bp in length with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 10 cycle PCR. Library concentration was quantified using a Qubit 2.0 fluorometer (Life Technologies). Insert size was checked on an Agilent 2100 and quantified using qPCR. Raw data were recorded in a FASTQ file. Raw reads were filtered to remove reads with adapter contamination or reads with low quality. Pair-ended clean reads were mapped with HISAT2 (HISAT version 2.1.0; Kim et al 2019) to the Phaseolus vulgaris reference genome (P.vulgaris v2.1, U.S. Department of Energy Joint Genome Institute, Phytozome v12.0: http://phytozome.jgi.doe.gov/).
SUB14711433
10DAA1