50 bp single end sequencing approach

The ‘Plant/Fungi Total RNA Purification Kit’ (Norgen Biotek Corp, ON, Canada) was used to isolate total RNA as per the manufacturer’s protocol. RNA quality and quantity were analyzed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA samples with a RIN number > 7.0 were used to prepare small RNA sequencing (sRNAseq) and messenger RNA sequencing (mRNAseq) libraries. For both sRNAseq and mRNAseq libraries, approximately 1 ug of total RNA were used. Illumina Hiseq 2500 sequencing platform and 50 bp single-end sequencing approach was used at LC Sciences (Houston, TX, USA) according to the vendor’s protocol [20 (link)]. For mRNAseq libraries, ribosomal RNA depletion was performed following the protocol described in the Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, CA, USA). Oligo-(dT) magnetic beads were used to purify poly(A) mRNA. Using a divalent cation buffer under elevated temperature, poly(A) RNA was fragmented. This procedure was followed by reverse-transcription of cleaved mRNA fragments to produce cDNA that was subsequently used to produce U-labeled second strand DNA. Following end repair, 3′ adenylation, adapter ligation, and PCR, final libraries were made. Bioanalyzer 2100 was used for quantification and quality control of the mRNAseq libraries. Illumina’s NovaSeq 6000 sequencing platform was used to perform paired-end (150 bp) sequencing.

The ‘Plant/Fungi Total RNA Purification Kit’ (Norgen Biotek Corp, ON, Canada) was used to isolate total RNA as per the manufacturer’s protocol. RNA quality and quantity were analyzed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA samples with a RIN number > 7.0 were used to prepare small RNA sequencing (sRNAseq) and messenger RNA sequencing (mRNAseq) libraries. For both sRNAseq and mRNAseq libraries, approximately 1 ug of total RNA were used. Illumina Hiseq 2500 sequencing platform and 50 bp single-end sequencing approach was used at LC Sciences (Houston, TX, USA) according to the vendor’s protocol [20 (link)]. For mRNAseq libraries, ribosomal RNA depletion was performed following the protocol described in the Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, CA, USA). Oligo-(dT) magnetic beads were used to purify poly(A) mRNA. Using a divalent cation buffer under elevated temperature, poly(A) RNA was fragmented. This procedure was followed by reverse-transcription of cleaved mRNA fragments to produce cDNA that was subsequently used to produce U-labeled second strand DNA. Following end repair, 3′ adenylation, adapter ligation, and PCR, final libraries were made. Bioanalyzer 2100 was used for quantification and quality control of the mRNAseq libraries. Illumina’s NovaSeq 6000 sequencing platform was used to perform paired-end (150 bp) sequencing.