MeRIP-Seq was performed according to the published procedure (42 (link)). Briefly, fragmented mRNA was incubated with anti-m6A polyclonal antibody (Synaptic Systems, 202003) in immunoprecipitation buffer for 2 h at 4 °C and then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) for another 2 h at 4 °C. Bound RNA was eluted from the beads with m6A (BERRY & ASSOCIATES) and extracted with the TRIzol reagent (Invitrogen). Fragmented mRNA without immunoprecipitation was used as input control. RNA-seq libraries were generated with the Next® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs). Sequencing data were obtained from the HiSeq 2500 system (Illumina). After quality control, clean data were aligned to the reference genome (UCSC mm10) with Hisat2 software v2.2.1 (71 (link)). The read alignment on the genome was visualized by using the IGV tool v2.14.0 (74 (link)). MACS software v3.0.0a7 (75 (link)) was used for m6A peak calling with the significance cutoff q-value < 0.05. Peaks were annotated as located in 5′-UTR, CDS, 3′-UTR, intronic region and intergenic region. The metagene profile was drawn by the R package Guitar v2.12.0 (76 (link)). Motifs in m6A peaks were identified using HOMER v4.7 (77 (link)).
Next ultra 2 directional rna library prep kit
Manufactured by New England Biolabs
Sourced in United States
The Next Ultra II Directional RNA Library Prep Kit is a reagent kit designed for the preparation of directional RNA sequencing libraries. The kit provides reagents and protocols for reverse transcription, second-strand synthesis, adapter ligation, and PCR amplification of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500 system, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Protein a bead, by Thermo Fisher Scientific (1 mentions)
Anti m6a polyclonal antibody, by Synaptic Systems (1 mentions)
Protein a magnetic beads, by Thermo Fisher Scientific (1 mentions)
Hiseq sequencer, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnaqueous micro kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
5 protocols using next ultra 2 directional rna library prep kit
1
N6-methyladenosine Mapping by MeRIP-Seq
2
RNA-seq Library Preparation and Sequencing
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The RNA libraries were prepared using NEB Next Ultra II Directional RNA Library Prep Kit at University of California Riverside (UCR) genomics core. The RNA libraries were analyzed by the bioanalyzer. Low quality libraries were removed from the samples. At least three biological replicates for each data point were sequenced. A total of 17 libraries (3 each for W0 and W6, 6 for W4, and 5 for WB Table S1 ) were pooled and sequenced using a 1 × 75 × 6 cycle NextSeq v2 high output run. A sample tree was created using Spearman correlation. Biological replicates from the same time points clustered together and all time points had their own branch, which confirms the reproducibility of our results (Figure S1 ). The raw reads data was deposited to the NCBI’s Sequence Read Archive (SRA) accession # SRP218030, and BioProject # PRJNA559688.
3
Profiling m1A RNA Modifications
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Fragmented RNA was incubated with an anti-m1A polyclonal antibody (MBL, Japan) in an immunoprecipitation buffer for 2 h at 4°C. The reaction mixture was then immunoprecipitated with protein A magnetic beads (Thermo Fisher) at 4°C for 2 h. Next, the bound RNA was eluted from the beads with m1A antibody in IPP buffer and extracted with the TRIzol reagent. Extracted RNA was prepared using the NEB Next Ultra II Directional RNA Library Prep Kit (NEB, United States). Both the input sample (without immunoprecipitation) and the m1A antibody-immunoprecipitated samples were subjected to 150 bp paired-end sequencing on an Illumina HiSeq sequencer.
4
RNA-Seq Analysis of Offspring Tissues
5
Synchronization and Transcriptome Analysis of C. elegans
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CX12311, PTM413, and PTM414 were synchronized using alkaline-bleach to isolate embryos, which were washed with M9 buffer and placed on a tube roller overnight. Approximately 400 hatched L1 animals were placed on NGM agar plates for each strain and incubated at 20°C for 48 hours. The ~L4 stage animals were washed off for standard RNA isolation using Trizol. Four replicates for each strain were performed on different days. The RNA libraries were prepared using the NEB Next Ultra II Directional RNA Library Prep Kit (E7760S) following its standard protocol. The libraries were sequenced by Illumina NextSeq 500. The reads were aligned by HISAT2 using default parameters for pair-end sequencing. Transcript abundance was calculated using HTseq and then used as inputs for the SARTools [88 (link), 89 (link)]. edgeR v3.16.5 was used for normalization and differential analysis[55[90 ]. The analysis result was shown in a volcano plot. CX12311 was treated as the wild type. The genes show significant differential expression in the volcano plot are under thresholds | log2(fold) | > 1 and FDR adjusted p-value < 0.01. Sequencing reads were uploaded to the SRA under BioProject PRJNA526525.
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