Truseq paired end cluster kit

cDNA libraries were constructed from 18 leaf and two floret samples using a TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) following the manufacturer’s instructions. Library quality was confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and quantified via quantitative real-time PCR (qPCR) (Illumina protocol SY-930-10-10). Clustering was performed using a TruSeq Paired-End Cluster Kit on a cBot (Illumina Inc.). Paired-end sequencing was performed on an Illumina Genome Analyzer IIx Platform with TruSeq SBS 36-Cycle kits (Illumina Inc.) following the manufacturer’s specifications. RNA-seq was performed with floret libraries in two separate lanes without biological replicates. The remaining 18 leaf libraries from six different accessions (Table 1), each with three biological replicates, were distributed randomly in the flow cell with three libraries per lane.
The raw data was converted to FastQ files containing 72-bp reads. Quality control was performed using the NGS QC Toolkit v2.3.3 [52 (link)]. Initially, high-quality reads (Phred quality score ≥ 20 in at least 75% of bases) and reads with more than 60 bases were selected. Subsequently, reads were trimmed at the 3′ end for the removal of barcodes.

Directional bisulfite-converted libraries for paired-end sequencing were prepared using the Ovation Ultralow Methyl-Seq Library System (NuGen). The manufacturer’s suggested protocol was followed. Briefly, this entailed fragmentation, end repair, adapter ligation, final repair, bisulfite conversion, and PCR amplification. We used 27, 14, and 33 ng of DNA for H, DFS, and MBC, respectively, in 50 μl T low E buffer, which was fragmented to an average size of 200 bp using the Covaris S2 system (Additional file 3: Figure S2A). Bisulfite conversion was performed using the EpiTect Fast DNA Bisulfite Kit (Qiagen) as per manufacturer’s instructions. Post-library QC was performed with BioAnalyzer DNA 1000 chips (Agilent) and the Qubit dsDNA High Sensitivity fluorometric assay (Invitrogen). An equimolar pool of the prepared libraries was created at a concentration of 5 nM. The sample was subsequently diluted and clustered on the Illumina cBot using TruSeq Paired End Cluster Kit v.3 chemistry. Paired-end sequencing was performed on the Illumina HiSeq 2500 platform using TruSeq SBS v3 kits for a total read length of 200 bp.