Truseq stranded mrna lt sample prep protocol

Before cDNA construction, the RNA integrity number (RIN) was measured in Bioanalyzer (Agilent Bioanalyzer 2100 system, Agilent Technologies, Santa Clara, Calif, USA). The library preparation of 12 cDNA samples and Illumina sequencing were performed at the Laboratory of Functional Genomics Applied to Agriculture and Agri-Energy, University of São Paulo (USP), São Paulo, SP, Brazil. The Illumina TruSeq Stranded mRNA LT Sample Prep Protocol was used to cDNA libraries construction. The 12 libraries were sequenced using the HiSeq Flow Cell v4, with the Illumina HiSeq 2500, producing 125 bp paired-end reads (2×).

Tumorspheres treated with 100 nM (+)-JQ1 or 100 nM (−)-JQ1 were collected after 4 days of culture and the RNA was extracted using RNeasy Mini Kit (QIAGEN, UK). The RNA quality and quantity were assessed using automated capillary gel electrophoresis on a Bioanalyzer 2100 with RNA 6000 Nano Labchips according to the manufacturer’s instructions (Agilent Technologies, Ireland). Only samples that presented an RNA integrity number (RIN) higher than 8.0 were considered to the sequencing (Supplementary Table S5). RNA libraries were constructed using the TruSeq™ Stranded mRNA LT Sample Prep Protocol and sequenced on Illumina HiSeq. 2500 equipment in a HiSeq Flow Cell v4 using HiSeq SBS Kit v4 (2 × 100 pb).

The cDNA libraries were sequenced at the Center of Functional Genomics, ESALQ-USP (Piracicaba/Brazil). A total of 15 cDNA libraries were prepared using the Illumina TruSeq Stranded mRNA LT Sample Prep Protocol and paired-end sequenced using HiSeq SBS Kit v4 on the HiSeq2500 system (Illumina, San Diego, CA, USA). These libraries were deposited under access number GSE128577 at GenBank. The raw RNA-seq data was initially filtered by SeqyClean version 1.10.09 and checked for quality through FastQC software v. 0.11.8. The alignment of the reads against the papaya reference genome, available at NCBI (Papaya1.0, GCF_000150535.2), was performed by the software STAR v. 2.6.0a to obtain the uniquely aligned reads [19 (link),25 ,26 ,27 (link)]. The BlastX tool was used to align the extracted transcript sequences to Viridiplantae (txid33090) non-redundant (nr) database.