Indexes 1 12

Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., Hitchin, UK). mRNA was isolated using a Poly(A) RNA Selection Kit (LEXOGEN, Inc., Wien, Austria). The isolated mRNAs were used for cDNA synthesis and shearing following the manufacturer’s instructions. Indexing was performed using Illumina indexes 1–12. Enrichment was performed using PCR. Subsequently, libraries were examined using a TapeStation HS D1000 Screen Tape (Agilent Technologies, Amstelveen, The Netherlands) to evaluate the mean fragment size. Quantification was performed using a library quantification kit and StepOne Real-Time PCR System (Life Technologies, Inc., Carlsbad, CA, USA). High-throughput sequencing was performed via paired-end 100 sequencing using a NovaSeq 6000 (Illumina, Inc., San Diego, CA, USA).

Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK). The isolation of mRNA was performed using the Poly(A) RNA Selection Kit (LEXOGEN, Inc., Austria). The isolated mRNAs were used for the cDNA synthesis and shearing, following manufacture’s instruction. Indexing was performed using the Illumina indexes 1–12. The enrichment step was carried out using of PCR. Subsequently, libraries were checked using the TapeStation HS D1000 Screen Tape (Agilent Technologies, Amstelveen, The Netherlands) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA).
A quality control of raw sequencing data was performed using FastQC [33 ]. Adapter and low quality reads (< Q20) were removed using FASTX_Trimmer and BBMap [34 , 35 ]. Then the trimmed reads were mapped to the reference genome using TopHat [36 (link)]. The RC (Read Count) data were processed based on FPKM + Geometric normalization method using EdgeR within R [37 ]. FPKM (Fragments Per kb per Million reads) values were estimated using Cufflinks [38 (link)].

Libraries were prepared from 3 μg of total RNA using the SMARTer Stranded RNA-Seq kit (Clontech). The isolation of mRNAs was performed using the Illumina beads and the TruSeq protocol (Illumina). The final elution of the mRNAs was performed using the Illumina elution buffer (19.5 μl). The isolated mRNAs were quantified using a Qubit fluorometer, as described above. Ten nanograms of mRNA were used for the cDNA synthesis and shearing, following the manufacturer’s instruction. Indexing was performed using the Illumina indexes 1–12. The enrichment step was carried out using 12 cycles of PCR. Subsequently, libraries were checked using a 2100 Bioanalyzer (DNA High sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the KAPA library quantification kit (KAPA Biosystems) using a ViiA7 Real-Time PCR System (Life Technologies). The pooled libraries (20 pM) were sequenced on an Illumina MiSeq in six consecutive runs (MiSeq reagent kit V3, 150 cycles) generating 76 base pairs (bps) paired-end reads. Raw sequences have been deposited at the NCBI Gene Expression Omnibus (GEO), http://www.ncbi.nlm.nih.gov/geo, accession number: GSE85144.