An RNeasy kit (Qiagen) was used to isolate RNA, and the Ribo-Zero magnetic kit (epicenter) was used to deplete rRNA. The NEBNext mRNA Library prep kit for Illumina (NEB) was used to prepare cDNA libraries for sequencing. Libraries were sequenced on an Illumina HiSeq2500 with 15 million read sequencing depth at the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory. To analyze the data, Trimmomatic version 0.32 (83 (link)) was used to trim the low-quality bases from the reads, with parameters set to LEADING:27 TRAILING:27 SLIDINGWINDOW:4:20 MINLEN:35. The trimmed reads were mapped to the genome using Bowtie 1.0.1 (84 (link)). SAMtools 0.1.19 (85 (link)) was used to sort the mapped reads. Read counts per gene or transcriptional unit were calculated using easyRNASeq (86 (link)), using .gff gene description files generated from the curated genome hosted by NCBI (NC_008463.1 ) (see Dataset S2 ), modified by the results of the single nucleotide resolution sequencing published by Wurtzel et al. (46 (link)) (see Dataset S3 ). Significance values for differential expression were determined (47 (link)) using Degust. The full data set is publicly available through the NCBI gene expression omnibus (GEO GSE65393 ).
Nebnext mrna library prep kit
Manufactured by Illumina
The NEBNext mRNA Library Prep kit is a laboratory equipment product designed for preparing mRNA samples for sequencing. It includes the necessary reagents and protocols to extract, purify, and convert mRNA into a sequencing-ready library. The kit's core function is to enable the analysis of gene expression profiles by generating cDNA libraries from mRNA samples.
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Lab products found in correlation
2 protocols using nebnext mrna library prep kit
1
RNA-seq Protocol for Differential Expression
2
RNA-seq analysis of mouse brain regions
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A total of 64 samples were used in the study. The hippocampus and cortex were dissected from the left hemisphere of each mouse brain and snap frozen in liquid nitrogen. Tissues were homogenized in Qiazol reagent and total RNA isolated using the RNeasy Plus Universal Mini Kit according to the manufacturer's protocol (Qiagen). RNA integrity was measured for all samples using the Bioanalyser Agilent 2100 series using RNA nano chip. All sequencing libraries analyzed were generated from RNA samples measuring an RNA integrity score ≥8.9. The Illumina TruSeq mRNA stranded protocol was used to obtain poly-A mRNA from all samples. Hundred nanogram of isolated mRNA was used to construct RNA-seq libraries. Libraries were quantified and normalized using the NEBNext mRNA Library prep kit for Illumina and sequenced as paired-end 76 bp reads on the Illumina NextSeq 500 platform using technical replicates.
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