Truseq stranded rna sample preparation kit

TruSeq Stranded RNA-seq libraries (TruSeq Stranded RNA Sample Preparation Kit, Illumina, San Diego, CA, USA) were produced from high-quality RNA (1 μg) by the HTS lab (University of Illinois, Urbana, IL, USA) following standard protocols. Qubit (Life Technologies, Carlsbad, CA, USA) was used to quantify libraries, and an Agilent bioanalyzer DNA7500 DNA chip (Agilent Technologies, Wilmington, DE, USA) was used to determine the average library size, which was diluted to 10 nM. Finally, library dilutions were quantitated using an ABI 1900 to ensure high accuracy quantification for consistent pooling and cluster maximization on the Illumina flowcell.

All RNA analytes were assayed for RNA integrity, concentration, and fragment size. Samples for total RNA-Seq were quantified on a TapeStation system (Agilent, Inc. Santa Clara, CA, USA). RNA Integrity score (RIN) averaged at 7.4. Samples with RINs > 7.0 were considered high quality. Total RNA-Seq library construction was performed from the RNA samples using the TruSeq Stranded RNA Sample Preparation Kit and bar-coded with individual tags following the manufacturer's instructions (Illumina, Inc. San Diego, CA). Libraries were prepared on an Agilent Bravo Automated Liquid Handling System. Quality control was performed at every step and the libraries were quantified using a TapeStation system. Indexed libraries were prepared and run on the NovaSeq 6000 to generate an average of 35 million reads per sample. The raw Illumina sequence data were demultiplexed and converted to FASTSQ files, and adapter and low-quality sequences were quantified.

Retinae were dissected from the RNAlater-stored eyes of each of the three sampled stomiids (two M. niger (MN1, MN2) and a single P. microdon (PM1)). Retina tissue-specific total RNA was extracted from each individual separately, using the RNeasy^® Mini Kit (Qiagen, Valencia, California) standard protocol, including DNase purification. Quantity and quality of extracted RNA was assayed for each sample using an Agilent 2100 Bioanalyzer with RNA6000 Nano chip kit (Agilent Technologies, Inc., Santa Clara, California). Bar-coded, strand-specific cDNA libraries were prepared for each individual using the TruSeq^® Stranded RNA sample preparation kit (Illumina, San Diego, California), according to manufacturer’s instructions. Ribo-Zero Gold rRNA depletion was carried out prior to cDNA synthesis. Individual cDNA libraries were diluted to 2 nM, and 5 μl from each library was pooled for multiplexing. 6 pM of this pooled sample was added to each of three flow cell lanes on an Illumina HiSeq 2500 sequencer, generating 100 bp paired-end reads. Read quality trimming and sequencing adapter removal were carried out using Trimmomatic v3.342 (link). Using a sliding window of 5 bp, reads were trimmed where the average Phred quality score dropped below 30. Reads below a minimum length of 50 bp were removed.

In total, 23 cDNA libraries were constructed using an Illumina Truseq stranded RNA sample preparation kit following the manufacturer’s instruction (Illumina Inc.; San Diego, CA). Briefly, polyA containing mRNA molecules were purified by oligo (dT) magnetic beads and subsequently fragmented. The purified RNA fragments were reversely transcribed into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen™; Austin, TX). The second-strand cDNA was synthesized using dUTP instead of dTTP, as a result, the second-strand cDNA was not amplified during PCR because the polymerase can’t add nucleotide to dUTP. The double-strand cDNA was adenylated at the 3’ end and ligated to the Illumina indexing adapters. After PCR enrichment, cDNA quantity and quality were assessed using a NanoDrop ND-1000 spectrophotometer and Agilent 2100 bioanalyzer. The averaged size of synthesized cDNA fragments was approximately 260 bp. cDNA libraries were normalized to 10 nm for each sample and then pooled together and sequenced on four lanes of an Illumina Hiseq 2000 sequencer at Delaware biotechnology institute, University of Delaware. Approximate 68 million fragments per sample were sequenced by 75-bp from both ends.

RIP assays were performed with the Magna RIP RNA‐binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA), according to the manufacturer's instructions. In brief, SH‐SY5Y cells were harvested in RIP lysis buffer, then incubated overnight at 4 °C with gentle rotation, with 7 µg SHMT2 antibody (Genetex) or an equal volume of control IgG that was conjugated to magnetic beads. Bioanalyzer 2100 system (Agilent) was used for RNA quality control. TruSeq Stranded RNA Sample Preparation Kit (Illumina) was used to construct the RNA library. High‐throughput sequencing was performed on IlluminaHiSeq 2500 by Seqhealth (Wuhan, China). Clean reads were normalized to calculate Per Kilo bases per Million reads (RPKM), the gene in which FPKM from IP compared to input was computed in a fold change > 2 and p‐value < 0.05 was chosen as significant enrichment. To analyze the GO analysis by using GOseq R package. To determine which gene is commonly bind but significantly altered in CTRL and KEN group, the genes with p < 0.05, |log2 Fold change| > 0.5 between 2 groups were selected to be analyzed. Motif identification was queried in MEME program (https://meme‐suite.org/meme/doc/meme.html). The search parameters were as follows: maximum width 12 amino acids, minimum motif width 4 amino acids, and other parameters were as defaults.