Nebnext ultra rna library preparation kit

High-quality total RNA (RIN > 7) from three independent experimental replicates of Patient-RPE, Control-RPE, Patient-fibroblasts, and Control-fibroblasts was extracted by RNeasy mini kit (Qiagen, Germany) and quantified by Agilent 2100 Bioanalyser. Total RNA (200 ng) was used to generate barcoded RNA-Seq libraries using the NEBNext Ultra RNA Library preparation kit (New England Biolabs). First, poly A + RNA was purified using poly-T oligo-attached magnetic beads followed by fragmentation and first and second cDNA strand synthesis. Next, cDNA ends were repaired and adenylated. The NEBNext adaptor was then ligated, followed by uracil excision from the adaptor and PCR amplification. The size of the libraries was checked using the Agilent 2100 Bioanalyzer and the concentration was determined using the Qubit^® fluorometer (Life Technologies). Libraries were sequenced on a HiSeq2500 (Illumina) to generate 60-base (sequencing batch 1) or 50-base (sequencing batch 2) single-end reads, to a mean depth of 100M reads/sample for one replicate for each condition (sequencing batch 1) and a mean depth of 20M reads/sample for the other two replicates (sequencing batch 2). FastQ files for each sample were obtained using CASAVA v1.8 software (Illumina).

RNA was extracted from bacterial cell pellet using Hi PURA Bacterial RNA Purification kit (HiMedia, USA) followed by measurement of concentration using QubIT and QC analysis using RNA 6000 Nano Bioanalyzer kit. Whole transcriptome library was prepared from QC qualified samples (RIN > 7) using NEB next ultra RNA library preparation kit (NEB). In brief, 10 μg of Total RNA was taken for ribosomal RNA depletion using Ribominus Bacteria kit module (Invitrogen Inc., USA). The rRNA depleted samples were fragmented using enzymatic method. First strand cDNA synthesis was done using random primers followed by second strand cDNA synthesis, end repair and adapter ligation. The adapter ligated libraries were multiplexed by adding index sequences via amplification. The adapter ligated and indexed libraries were quantitated by QubIT and validated by using Agilent HS kit. Resulting validated libraries were pooled in equimolar ratio and sequenced on NextSeq500 platform (Illumina, USA) using 2x150bp chemistry. The raw data was processed further after necessary quality check with an average Q30 > 70%. All the raw sequence data has been submitted to Sequence Read Archive. Relevant accession no. are SRX2855033 and SRX2855034.

Pectoral muscles from three pigeons of each breed from each stage (n = 3) were utilized. Total RNA was extracted using a Trizol kit (Invitrogen, Carlsbad, CA, USA) Manufacturer’s agreement. RNA quality was assessed on the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and RNase-free agarose gel was used for detection electrophoresis. After total RNA extraction, eukaryotic mRNA was enriched with Oligo (dT). The enriched mRNA was then fragmented into short fragments using fragmentation buffer and reverse transcribed into cDNA using the NEBNext Ultra RNA Library Preparation Kit (NEB#7530, New England Biolabs, Ipswich, MA, USA). End repair of purified double-stranded cDNA fragments was performed. A base was added, and connected to the Illumina sequencing adapter. The ligation reaction was purified with AMPure XP Beads (1.0×). Ligated fragment size was selected by agarose gel electrophoresis and polymerase chain reaction (PCR) amplified. The cDNA library was sequenced by Illumina Novaseq6000 Gene Denovo Biotechnology Co. (Guangzhou, China).

The MiniBEST Plant RNA Extraction Kit (TaKaRa) was used to extract total RNA. RNA concentration and quality were evaluated with the Agilent 2100 Bioanalyzer. A total of 45 RNA-seq libraries were created via NEB Next Ultra RNA Library Preparation Kit (NEB, E7530) and the NEB Next Multiplex Oligos (NEB, E7500). Next, paired-end sequencing was carried out using an Illumina Hiseq 2500 platform. The Trinity software was employed to compute the transcript abundance of genes in transcripts per million (TPM). Differentially expressed genes were identified with |log₂ (fold change) |≥ 1 and p-value (Padj) < 0.05, as detected by DESeq [20 (link)]. The RNA-seq data included in this study were obtained from Han et al. (2023).

We ground the colon tissues into powder and extracted total RNA using TRIzol (Takara Bio, Otsu, Japan) according to a previously described method [50 (link)]. The concentration and quality of total RNA were determined with a Nanodrop 1000 spectrophotometer. The absorbed optical density ratio (OD260/280) for RNA was selected to maintain a high RNA purity (1.85–2.05), and the RNA integrity was verified by 1.4% agarose-formaldehyde gel electrophoresis. The concentration of each RNA sample was normalized to 500 ng/μL for each sample based on optical density, and each sample was preserved at − 80 °C for subsequent analyses. Then, 1 μg of RNA was used for sequencing, sequencing libraries were generated using a NEBNext Ultra RNA Library Preparation Kit (E7530L, NEB, USA) following the manufacturer’s recommendations for Illumina Hiseq 2000 platform, and index codes were added to attribute sequences to each sample. Another 1 μg of RNA was reverse-transcribed using a PrimeScript® RT reagent Kit with a gDNA Eraser (Takara Bio, Shiga, Japan). The primer sets used in our research were listed in Additional file 10: Table S9.