Total RNA was isolated from DCs. Polyadenylated RNA was further enriched from total RNA by using Dynabeads® mRNA Purification Kit (Invitrogen). RNA samples were fragmented into ~100-nucleotide-long fragments with sonication. Fragmented RNA (100 ng mRNA or 5 μg total RNA) was performed m6A-IP following EpiMark N6-methyladenosine enrichment kit (NEB E1610S) protocol. RNA was enriched through RNA Clean&Concentration-5 (Zymo Research) and used for library generation with SMARTer Stranded Total RNA-Seq Kit (Takara). Sequencing was performed at the University of Chicago Genomics Facility on an Illumina HiSeq4000 machine in single-read mode with 50 bp per read. Sequencing reads were aligned to the mouse genome mm9 by STAR (version 2.6.0c)29 (link). The m6A-enriched regions (peaks) in each m6A-IP sample were detected by MACS2 (version 2.1.1.20160309)30 (link) with q value less than 0.01 and corresponding m6A-Input sample was used as the control. Peaks that were detected by both replicates were considered as high confident peaks. The peaks annotation and binding motif were analyzed by HOMER (version 4.9)31 (link).
Rna clean concentration 5
Manufactured by Zymo Research
The RNA Clean&Concentration-5 is a laboratory product designed to extract and concentrate RNA samples. It provides a simple and efficient method for purifying RNA from various sources, including cells, tissues, and bodily fluids. The product utilizes a unique binding matrix to selectively capture and purify RNA, while effectively removing contaminants and inhibitors. The concentrated RNA can then be eluted and used for downstream applications, such as reverse transcription and PCR analysis.
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2 protocols using rna clean concentration 5
1
m6A-seq Analysis of Murine Dendritic Cells
2
m6A-seq Analysis of Murine Dendritic Cells
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Total RNA was isolated from DCs. Polyadenylated RNA was further enriched from total RNA by using Dynabeads® mRNA Purification Kit (Invitrogen). RNA samples were fragmented into ~100-nucleotide-long fragments with sonication. Fragmented RNA (100 ng mRNA or 5 μg total RNA) was performed m6A-IP following EpiMark N6-methyladenosine enrichment kit (NEB E1610S) protocol. RNA was enriched through RNA Clean&Concentration-5 (Zymo Research) and used for library generation with SMARTer Stranded Total RNA-Seq Kit (Takara). Sequencing was performed at the University of Chicago Genomics Facility on an Illumina HiSeq4000 machine in single-read mode with 50 bp per read. Sequencing reads were aligned to the mouse genome mm9 by STAR (version 2.6.0c)29 (link). The m6A-enriched regions (peaks) in each m6A-IP sample were detected by MACS2 (version 2.1.1.20160309)30 (link) with q value less than 0.01 and corresponding m6A-Input sample was used as the control. Peaks that were detected by both replicates were considered as high confident peaks. The peaks annotation and binding motif were analyzed by HOMER (version 4.9)31 (link).
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