MeRIP was conducted as previously published with minor revisions.32 (link) Total RNA was evaluated with a NanoDrop ND-1000, and intact mRNA was isolated using an Arraystar Seq-Star™ poly(A) mRNA Isolation Kit (Arraystar, MD, USA) according to the manufacturer's instructions. Purified mRNA was randomly fragmented into approximately 100-nt fragments by incubation in fragmentation buffer. Fragmented mRNA was immunoprecipitated with anti-m6A antibody (202,003, Synaptic Systems), and 1/10 of the fragmented mRNA was kept as input for further RNA sequencing. RNA-seq libraries were prepared using the KAPA Stranded mRNA-seq Kit (Illumina, CA, USA). The methylated RNA was purified and sequenced on an Illumina HiSeq 4000 platform by Aksomics (Shanghai, China).
Kapa stranded mrna seq kit
Manufactured by Illumina
Sourced in United States
The KAPA Stranded mRNA-Seq Kit is a library preparation kit designed for sequencing mRNA transcripts. It enables the preparation of stranded mRNA libraries from total RNA samples for downstream next-generation sequencing analysis.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Hiseq 4000 system, by Illumina (3 mentions)
Anti m6a antibody, by Synaptic Systems (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Seq star poly a mrna isolation kit, by Arraystar (2 mentions)
Hiseq 3000, by Illumina (2 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Bp231, by Thermo Fisher Scientific (1 mentions)
Ercc spike in mix, by Thermo Fisher Scientific (1 mentions)
Ribo zero rrna removal kit, by Illumina (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Standard sensitivity ngs fragment analysis kit, by Agilent Technologies (1 mentions)
22 protocols using kapa stranded mrna seq kit
1
m6A-seq protocol for mRNA methylation
2
Epigenetic regulation in GP5d cells
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DNMT and HDAC inhibition in GP5d cells were performed as described earlier56 (link). GP5d cells were seeded in 6 well plates and treated with 500 nM/L 5-aza-2’-deoxycytidine (MedChemExpress, HY-A0004) or DMSO (Fisher, BP231). 5-aza-2’-deoxycytidine containing media were replaced each day for three days. SB939 was added at a concentration of 500 nmol/L (MedChemExpress, HY-13322) and cells were harvested after 18 hours. Total RNA was extracted using RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions in three biological replicates. RNA-seq libraries were prepared using 1 μg of total RNA input using KAPA stranded mRNA-seq kit for Illumina (Roche) as per manufacturer’s instruction and paired-end sequenced on NovaSeq 6000 (Illumina).
3
RNA-seq Protocol with ERCC Spike-Ins
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RNA was extracted using RNeasy Plus kits (QIAGEN) from 26 daughter clones (Table S2 ). To control for non-biological variation in expression data, ERCC Spike-In Mix (Thermo Fisher) was added (1ug total RNA spiked with 2uL of 1:100 ERCC mix). rRNA was depleted using Ribo-Zero rRNA Removal Kit (Illumina) and 150 base short-insert cDNA libraries generated using KAPA Stranded mRNA-Seq Kit (Illumina), following the balanced block design (Auer and Doerge, 2010 (link)). Sequencing (paired end, 75-bp read length) was performed on Illumina HiSeq 2500 platform. The sequencing reads were mapped using TopHat2 (Kim et al., 2013 (link)) (version 2.1.1) to the reference human genome (GRCh37) supplemented with control sequences to allow mapping of the Spike-In control RNA. Duplicate reads were removed using the Picard (MarkDuplicates) tool (version 1.60; https://broadinstitute.github.io/picard/ ). Gene expression levels were estimated with the default settings of the Cufflinks tool (Trapnell et al., 2010 (link)) (v.1.0.2), using a reference General Feature Format (GFF) file derived from Ensembl version 58. Only the high-confidence values (Cufflinks status ‘OK’) were considered in derivation of FPKM values and further analyses. A positive correlation between expected and observed burden of control Spike-In RNA implied the minimal technical variation affecting the data interpretation (not shown).
4
RNA-seq Analysis of Peritoneal Macrophages
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RNA from cell-sorted Peritoneal Macrophages (CD11b + F4/80 + CD19-) was extracted using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. Purified RNA samples were quantified using Qubit RNA HS Assay (Life Technologies, Thermo Fisher Scientific) and integrity was measured using the High Sensitivity RNA Analysis Kit (Advanced Analytical Technologies). Sequencing libraries were prepared using the KAPA Stranded mRNA-Seq kit according to the manufacturer's protocol (Illumina). The length of the libraries was determined by capillary electrophoresis using the Standard Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies). Libraries were quantified using the KAPA Library Quantification Kit (Kappa Biosystem) using the Eco PCR system (Illumina), following the manufacturer's protocol. Libraries were sequenced on a Miseq platform (Illumina) using a v3 150 kit with 2 × 75 bp paired-end. The subsequent analysis was performed in R using the Deseq2 package to normalize the data. Differentially expressed genes were identified using an adjusted p-value cut-off of 0.05 and a fold change of 1.5. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE167108.
5
RNA Extraction and RNA-Seq from ZIKV-Infected Trophoblasts
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Total RNA from hiPSC-derived trophoblast controls (mock) and trophoblasts infected for 96 h with ZIKVBR was extracted using the RNeasy Micro Kit(Qiagen, 74004), treated with TURBO DNase (Ambion, AM2238) for 30 min at 37°C, and then re-purified with the Qiagen RNeasy Micro Kit. RNA samples were quantified using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Q32852); purity was evaluated using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies) and the integrity was verified using the Agilent RNA 6000 Pico Kit (Agilent Technologies, 5067–1513) in the 2100 Bioanalyzer Instrument (Agilent Technologies). Stranded tagged cDNA libraries were prepared using the KAPA Stranded mRNA-Seq Kit (Illumina, KK8421) and cluster generation was performed using the Illumina HiSeq 4000 PE Cluster Kit (Illumina, PE-410-1001). Tagged libraries were pooled and sequenced (300 cycles, paired-end sequencing) in the Illumina HiSeq 4000 instrument using a HiSeq 4000 SBS Kit (Illumina, FC-410- 1003). Raw reads were preprocessed using the standard Illumina pipeline to segregate multiplexed reads.
6
Transcriptome Analysis of Mouse Tissue Samples
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At 11 weeks of age, six males of each group [SARA, EDMA, MANA, (SARAxMANA)F1, and (EDMAxMANA)F1] were sacrificed. Body weight, total body length, tail length, ear length, and hind foot length were measured, and liver and brown adipose tissue (BAT) were collected for RNA-sequencing. Further information for all mice used in this study can be found in S1 File . Liver and BAT were immediately placed in RNAlater, kept at 4°C for 24 hours and then stored at -80°C until RNA-extraction. We extracted RNA using the Qiagen RNeasy minikit following the manufacturer’s instructions. We validated RNA integrity using an Aligent Bioanalyzer and only RNA with RIN > 8 was used for library preparation. We generated cDNA libraries using the KAPA Stranded mRNA-Seq kit starting with 2ug of total RNA measured using Nanodrop, and uniquely indexed libraries using dual indexes from Illumina. We assessed library size and quality using an Aligent Bioanalyzer with a desired insert size of 300bp. Tissue-specific libraries were then pooled and sequenced across two lanes of Illumina 150bp paired-end NovaSeq, one using an S4 flow cell, and one using an S1 flow cell at the Vincent J. Coates Genomics Sequencing Center at UC Berkeley.
7
N6-Methyladenosine Transcriptome Profiling
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MeRIP and library preparation were conducted according to a previously published protocol with minor revisions [18 (link)]. One hundred twenty micrograms of purified total RNA was evaluated with a NanoDrop ND-1000. Intact mRNA was isolated from total RNA samples using an Arraystar Seq-Star™ poly (A) mRNA Isolation Kit (Arrarystar, MD, USA) following the manufacturer’s instructions and was chemically fragmented into 100-nucleotide-long fragments by incubation in fragmentation buffer. Fragmented mRNA was immunoprecipitated with anti-N6-methyladenosine (m6A) antibody (Synaptic Systems, Goettingen, Germany), and 1/10 of the fragmented mRNA was kept as input. RNA-seq libraries were prepared using a KAPA Stranded mRNA-seq Kit (Illumina, CA, USA). The completed libraries, qualified by an Agilent 2100 Bioanalyzer, were denatured and diluted to the loading volume of a loading concentration. Clustered libraries were loaded onto a reagent cartridge and forwarded for sequencing runs on an Illumina Hiseq 4000 system by Aksomics (Shanghai, China). Sequencing peaks were annotated to the Ensembl database. Sequence motifs were identified using MEME-ChIP analysis [19 (link)].
8
Comprehensive RNA Extraction and Sequencing
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Total RNA was extracted using miRNeasy Mini Kit (Qiagen, USA#217004, Germantown, MD, USA) and treated with DNase I using in-column DNase I digestion as described in the user’s manual. The integrity of RNA was checked using Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA samples with RNA integrity number (RIN) ~9.0 or more were used for further analyses. Poly (A) mRNA enrichment and construction of stranded mRNA-seq libraries were performed using KAPA Stranded mRNA-seq kit (Cat# KR0960) (Illumina, San Diego, CA, USA). Briefly, poly (A) mRNA was captured using magnetic oligo (dT) beads and fragmented using heat and magnesium. First-strand cDNA was synthesized using random primer and the second strand was synthesized in the presence of dUTP. Adenosine was added to the 3′-end followed by adaptor ligation. The library was amplified using high fidelity, low-bias PCR. Single-end sequencing of the libraries was done at the Sequencing and Analysis Core Resource, Duke University using Illumina Hi seq 2000 (Illumina, San Diego, CA, USA).
9
Transcriptomic Analysis of Plasmodium Parasites
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To compare the parasites’ transcriptomes upon stress conditions, we performed RNA-seq analysis using the ring-stage TetR-GCN5::GFP parasites with or without aTc. The experiment was done in two to three replicates. Total RNA was extracted using the ZYMO RNA Purification Kit, and RNA integrity was confirmed by the TapeStation system (Agilent). Total RNA was used to generate the sequencing libraries using the KAPA Stranded mRNA Seq Kit for the Illumina sequencing platform according to the manufacturer’s protocol (KAPA biosystems). Libraries were sequenced on an Illumina NextSeq 550 using 150 nt paired-end sequencing. Reads from Illumina sequencing were mapped to the P. falciparum genome sequence (Genedb v3.1) using HISAT2 (70 (link)). The expression levels and the differential expression were calculated by FeatureCounts and DESeq2 (71 (link), 72 (link)) with the criteria of ≥1.5-fold alteration and P-adj <0.1. RNA-seq data were submitted to the NCBI GEO repository (accession number GSE221211 ) with a token (cxmngauyrvcfjqj) for reviewers’ access.
10
Illumina Sequencing of Cellular RNA
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