Nebnext ultra 2 library prep kit

Cell samples at different stages were collected and mRNA-seq libraries were constructed with NEBNext® Ultra™ II Library Prep Kit for Illumina. Qualified libraries were multiplexed and sequenced on Illumina NovaSeq 6000 according to the manufacturer’s instructions (Illumina, USA). The Sequencing mode was PE150.
For data analysis, reference genome and gene annotation files were downloaded from GENECODE (hg38). Fastq files were pre-processed by Fastp (v0.12.1) (Chen et al. 2018 (link)) with default parameters. Cleaned data were then aligned to the reference genome via Hisat2 (v2.1.0) (Kim et al. 2015 (link)). FeatureCounts (v1.5.3) (Liao et al. 2014 (link)) was used to count the reads mapped to each gene.

Before library preparation, the integrity of each sample was assessed on an Agilent TapeStation system 4150 using RNA screen tape (Agilent Technologies Inc., Santa Clara, CA, USA). In contrast, RNA concentrations were measured using a Qubit 4 fluorometer (ThermoFisher Scientific Inc., Waltham, MA, USA). 100 nanograms of total RNA was used as input for ribosomal RNA (rRNA) depletion using the NEBNext rRNA depletion kit (New England BioLabs Inc., Ipswich, MA, USA, product code: E6350), according to the manufacturer’s instructions. Following rRNA depletion, total RNA libraries were built using the NEBNext Ultra II library prep kit from Illumina (New England BioLabs Inc., Ipswich, MA, USA, product code: E7775), according to the manufacturer’s instructions. The yield of amplified libraries was measured on a Qubit 4 fluorometer using a Qubit high-sensitivity DNA kit (HS DNA kit). Amplified libraries were further analyzed on HS D1000 screen tape on a TapeStation system 4150 to assess library size and molarity prior to pooling. The libraries were sequenced using Illumina HISeq-4000.

Control or HNRNPLi cells were harvested 4 days after siRNA transfection. Independent biological duplicates were obtained for both CTLi and HNRNPLi, and total RNA was isolated using the GeneJET RNA purification kit (Thermo Fisher Scientific: K0732) and quantified by Nanodrop. RNA-seq was performed using the Illumina (San Diego, CA) NovaSeq 6000. RNA-seq libraries were prepared with NEBNext Ultra II Library Prep Kit (Illumina (San Diego, CA): E7760) then multiplexed, and approximately 40 million reads per sample were obtained. Pair-end reads were aligned to the GENCODE v19 transcriptome hg19 using TopHat2 with default settings [55 (link)]. Differential expression among samples was calculated using ANOVA from the Partek Genomic Suite (Partek, St. Louis, MO). Analysis of the read count distribution indicated that a threshold of 10 reads per gene generally separated expressed from unexpressed genes, so all genes with fewer than 10 reads were excluded from ANOVA analysis. Gene lists for significantly up-regulated or down-regulated genes were created using p-value < 0.05 and ≥2-fold change. Enriched GO terms for RNA-seq differentially expressed gene sets were identified using Enrichr [56 (link),57 (link)]. Heatmaps for the RNA-seq data were generated using Partek Genomic Suite (http://www.partek.com/partek-genomics-suite/).