Nebnext ultra 2 rna library prep kit for

RNA was purified with the RNeasy Plus Micro Kit according to the manufacturer’s protocol (QIAGEN). RNA was quantified with a Qubit 2.0 fluorometer (Invitrogen) and the quality was assessed on a Bioanalyzer 2100 (Agilent) using a RNA 6000 Pico chip (Agilent). Samples with an RNA integrity number (RIN) of > 8 were used for library preparation. Barcoded mRNA-seq cDNA libraries were prepared from 10ng of total RNA using NEBNext® Poly(A) mRNA Magnetic Isolation Module and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® according to the manual with a final amplification of 15 PCR cycles. Quantity was assessed using Invitrogen’s Qubit HS assay kit and library size was determined using Agilent’s 2100 Bioanalyzer HS DNA assay. Barcoded RNA-Seq libraries were onboard clustered using HiSeq® Rapid SR Cluster Kit v2 using 8pM and 59bps were sequenced on the Illumina HiSeq2500 using HiSeq® Rapid SBS Kit v2 (59 Cycle). The raw output data of the HiSeq was preprocessed according to the Illumina standard protocol. Sequence reads were trimmed for adapter sequences and further processed using Qiagen’s software CLC Genomics Workbench (v12 with CLC’s default settings for RNA-Seq analysis). Reads were aligned to GRCm38 genome. Heatmaps were generated using the online tool Morpheus https://software.broadinstitute.org/morpheus.

Purified RNA was sent to Novogene (Sacramento, CA) for library preparation. Libraries were prepared from mRNA purified from total RNA using poly-T oligo-attached magnetic beads (NEBNext Ultra II RNA Library Prep kit for Illumina, New England Biolabs, E7775). Non-stranded library preparation was carried out using the NEBNext Ultra II RNA Library Prep kit for Illumina according to manufacturer protocol. Libraries were subsequently sequenced on a Novaseq 6000 S4 flow cell. 20 million paired-end reads (PE150) were generated for each sample.

Total RNA was extracted with the RNAprep Pure Plant Kit (TIANGEN, China) and then quantified using a Nanodrop2000 spectrophotometer (NanoDrop Technologies, USA) and an Agilent 2100 Bioanalyzer (Agilent, USA).
For Illumina RNA sequencing, sequencing libraries were generated from the total RNA samples with the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations. The cDNA libraries were sequenced on an Illumina HiSeq 2000 platform (Illumina, USA).

The RNA library was prepared by Illumina Ribo-Zero Plus rRNA Depletion Kit followed by NEBNext® Ultra™ II RNA Library Prep Kit for Illumina®. The sequencing was performed on Illumina Novaseq 6000 S4 Flowcell (PE150). The minimum coverage per sample is 40M Read Pairs (12G). All samples passed the quality control (QC).

RNA library preparation and sequencing were conducted at GENEWIZ, LLC (South Plainfield, NJ, USA). The cDNA sequencing libraries were prepared using NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra II RNA Library Prep Kit for Illumina, following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). The sequencing libraries were validated on the Agilent TapeStation System (Agilent Technologies, Palo Alto, CA, USA), and quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were sequenced as 2 × 150 paired-end (PE) using the Illumina NovaSeq Sequencing System. After sequencing, Trimmomatic (version 0.39) was used to remove adapters and filter the raw reads with < 35 bases, as well as the leading and trailing bases with quality < 20. The filtered reads were mapped to the sequence of the mouse genome (GRCm38) using HISAT2 (v2.1.0). Raw counts for each gene were obtained using featureCounts software included with the Subread package with the default option (-p -t exon). RUVSeq (version 1.18.0) was used for further normalization to account for sample variations.

Total RNA was extracted as described above. RNA-seq was conducted by Novogene. The library was generated with NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB; 7770), and sequencing was done using the NovaSeq 6000 S4 platform with PE150. The data were analyzed using Partek Flow (Version 10.0). After primary quality assessment was performed, bases and reads with low quality were filtered out and the reads were aligned to mouse reference genome (mm10) using the STAR (Version 2.7.8a).^{72 (link)} The final BAM files were quantified using the Partek E/M algorithm.^{69 (link)} Normalization of read count was performed by the total number of counts (count per million) plus 0.0001, and all genes with less than ten normalized read counts were excluded from subsequent analyses. Differentially expressed genes were identified using Partek gene-specific analysis (GSA) algorithm. Geneotology^{73 (link)} and Morpheus (RRID: SCR_017386) were used for data analysis.