For human iPSCs and ESCs, RNA sequencing libraries were prepared using Ribo-Zero rRNA Removal Kit (H/M/R) (Illumina, MRZH11124) and NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina (E7420). The total RNA input amount for the RiboZero kit was 1 μg total. The rRNA input for library construction was 50 ng total. For neural, muscle progenitors and DDX6 OE samples, RNA-seq libraries were constructed from polyadenosine (polyA)-selected RNA using NEBNext Ultra Directional RNA library prep kit for Illumina (New England BioLabs). Libraries were amplified for 14 cycles. Post library constructions, the samples were validated using 2200 Tapestation System and High Sensitivity D1000 ScreenTape kit. Libraries were quantified using the Kapa Biosystems Library Quantification kit (KK4828) and the BioRad CFX96 instrument. Each lane of sequencing was pooled into a 19-plex (19 samples per lane) with unique barcodes. Pooled libraries are also quantified using the Kapa Biosystems Library Quantification kit (KK4828) and the BioRad CFX96 instrument. These pools are then denatured to 16 pM with 1% PhiX and sequenced on the Illumina HiSeq2000 instrument, resulting in approximately 40 million reads per sample.
Nebnext ultra directional rna library prep kit
Manufactured by Illumina
Sourced in United States
The NEBNext Ultra Directional RNA Library Prep Kit is a laboratory equipment product. It is designed for the preparation of directional RNA-sequencing libraries from total RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (27 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (21 mentions)
Trizol, by Thermo Fisher Scientific (19 mentions)
Hiseq 2500, by Illumina (18 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (15 mentions)
Rneasy mini kit, by Qiagen (14 mentions)
Hiseq 4000 platform, by Illumina (14 mentions)
Ribo zero rrna removal kit, by Illumina (13 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (11 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (10 mentions)
Hiseq 2000, by Illumina (9 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (9 mentions)
Hiseq 2500 platform, by Illumina (8 mentions)
Hiseq platform, by Illumina (8 mentions)
Nextseq 500, by Illumina (7 mentions)
Hiseq 4000, by Illumina (7 mentions)
Epicentre ribo zero rrna removal kit, by Illumina (6 mentions)
Novaseq 6000, by Illumina (6 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (6 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (6 mentions)
203 protocols using nebnext ultra directional rna library prep kit
1
RNA-seq Library Preparation for iPSCs and ESCs
2
NEBNext Ultra Directional RNA Library Prep
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The NEBNext Ultra Directional RNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to process 38 samples (22 WM and 16 GM) (GenomeScan, Leiden, The Netherlands). The sample preparation was performed according to the protocol of NEBNext Ultra Directional RNA Library Prep Kit from Illumina (NEB #E7420). Briefly, mRNA was isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed with an input of 50–100 ng RNA. This cDNA was used for ligation with the sequencing adapters and PCR amplification of the resulting product. The quality (RIN) and yield after sample preparation was measured with the Fragment Analyzer. Thirty-one samples (21 WM and 10 GM) met the quality criteria and were selected for sequencing. The size of the resulting products was consistent with the expected size distribution (a broad peak between 400 and 700 bp). Clustering and DNA sequencing using the Illumina NextSeq500 SR75 was performed according to the manufacturer’s protocols. A concentration of 1.5 pM of DNA was used. At least 1.9 Gb (25 million SR75 reads) were generated per sample with a quality score of ≥30. NextSeq control software v1/4/8 was used. Image analysis, base calling, and quality check was performed with the Illumina data analysis pipeline RTA v2.4.6 and Bcl2fastq v2.17.
3
Transcriptome Analysis of Bxb1, prime, and PASTE
4
Transcriptome Profiling of GTEx and Neuromuscular Disorders
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FASTQs were downloaded from the Database of Genotypes and Phenotypes (dbGaP) under the project accessions phs000424.v8.p2 and phs000655.v3.p1.c1 for GTEx control individuals and neuromuscular disease patients, respectively. GTEx controls were selected for LCLs (n = 91), skeletal muscle (n = 184) and whole blood .
Depletion and NEBNext Ultra Directional RNA Library Prep Kits on a HiSeq 2000 instrument (Illumina). RNA samples from 20 LCLs were obtained from the kConFab consortium. Poly(A)-selected RNA was generated using the TruSeq Stranded mRNA Library Prep Kit (Illumina), and 150bp paired end reads created using the NextSeq 500 instrument (Illumina).
Depletion and NEBNext Ultra Directional RNA Library Prep Kits on a HiSeq 2000 instrument (Illumina). RNA samples from 20 LCLs were obtained from the kConFab consortium. Poly(A)-selected RNA was generated using the TruSeq Stranded mRNA Library Prep Kit (Illumina), and 150bp paired end reads created using the NextSeq 500 instrument (Illumina).
5
RNA-Seq of BETp and mTORi in CCA
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Prior to RNA or DNA extraction, viable cells were counted by mixing 10 μL of trypan blue (MilliporeSigma, 15250061) with 10 μL of cells. Experiments were carried out only when cell viability was > 90%. Total RNA from each group (vehicle, BETp, mTORi, and BETp + mTORi) of CCA cell lines (SNU1079 and SSP25) with 48 hours of drug treatment was isolated (Thermo Fisher Scientific, 12183025) and quality controlled with protocols from Illumina. A total amount of 2 μg RNA per sample was used as input material for library construction. Strand-specific sequencing libraries were generated through the dUTP method by using the resulting RNA from NEBNext Ultra Directional RNA Library Prep Kit for Illumina. RNA-Seq was performed on an Illumina HiSeq 2000 platform, and 100 bp paired-end reads were generated. In mRNA-Seq analysis, differential RNA expression patterns are identified through DESeq2 and EdgeR algorithms (42 (link), 43 (link)). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conduct by WebGestalt (44 (link)).
6
H. pylori Biofilm Transcriptomics
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Biofilms of H. pylori SS1 were grown in 6-well plates (Costar) in BB2 as described above. After 3 days of incubation, medium containing nonattached planktonic bacteria (the planktonic fraction) was removed by pipetting, and the cells were harvested by centrifugation and washed twice with PBS. The attached bacteria, representing the biofilm fraction, were washed twice with PBS to remove any remaining planktonic cells. Attached cells were scrapped off the plate using a cell scraper. Both planktonic and biofilm fractions were subject to total RNA extraction using the TRIzol Max bacterial enhancement kit (Ambion, Life Technology, Carlsbad, CA) as described by the manufacturer. RNA was further purified and concentrated using an RNeasy kit (Qiagen). rRNA was removed using the RiboZero magnetic kit (Illumina). Sequencing libraries were generated using NEBNext Ultra Directional RNA library prep kit for Illumina (NEB, USA). cDNA library quality and amount were verified using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA) and then sequenced using Illumina NextSeq Mid-Output (University of California Davis Genome Center).
7
Transcriptome Analysis of Pepper Tissue
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The transcriptome sequencings were commissioned from Novogene Technology Co. (Beijing, China). Total RNA was extracted from frozen pepper tissue using standard extraction methods, the RNA samples were rigorously quality controlled using the Agilent 2100 bioanalyzer, libraries were constructed using the NEBNext Ultra directional RNA library prep kit for illumina, and the mRNA libraries from each sample were subse-quently sequenced using Illumina. The low-quality reads, N-containing, and spliced reads were removed from the sequenced data. The Q20, Q30 and GC contents of the clean data were subsequently calculated. HISAT2 was used to map the paired-end clean reads to the reference genome for comparison. Novel gene prediction was performed using StringTie (1.3.3b) [51 (link)]. Differential expression analysis was performed using DESeq2 software (1.20.0) [52 (link)] to compare between combinations (genes with a p-value < 0.05 were assigned as differentially expressed) and GO and KEGG enrichment analysis was implemented.
8
RNA-seq Library Preparation for Illumina
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Sequencing libraries were prepared using NEB Next Ultra™ directional RNA Library Prep Kit for Illumina (San Diego, CA, USA) according to the manufacturer’s recommendations. Briefly, purification of mRNA was achieved from total RNA by using ploy-T oligo-attached magnetic beads. Then, adding the Illumina proprietary fragmentation buffer to the RNA samples, fragmentation of mRNA was completed under elevated temperature. Taking those fragments as templates, the first-strand cDNA was synthesized via using random hexamers and SuperScript II. The second-strand cDNA was synthesized by adding buffer, dNTPs, DNA polymerase I and RNase H to the reaction system. Subsequently, purification of the double-strand cDNA was performed using AMPure XP beads. The remaining overhangs were repaired via exonuclease/polymerase. Adenylation of the 3’ ends of cDNA fragments were conducted. Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. 200-bp cDNA fragments were extracted using an AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments were amplified by 12 cycles of PCR. After enrichment of DNA, the libraries were sequenced using Illumina Hiseq 2000 platform and raw data was generated. Adaptor, duplication and low quality sequences were removed to get clean data.
9
RNA-Seq Analysis of Fungal Isolates
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Total RNA of each isolate was extracted from mycelia using an RNAiso Plus kit (TaKaRa, China) according to the manufacturer’s instructions and then treated with DNase I. The concentration of each RNA extraction was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Total RNA was stored at −80 °C until being used for RNA-Seq analysis. Equal amounts of total RNA from 100 isolates collected annually were pooled in a single library according to the collection year. Three RNA libraries were generated and used for sequence analysis. All library preparations and sequencing steps were performed by GENEWIZ Inc. (China). rRNA was depleted from total RNA using the Illumina Ribo Zero rRNA removal Kit (Plant Leaf). Three libraries were constructed according to the manufacturer’s protocol (NEBNext Ultra Directional RNA Library Prep Kit for Illumina). Paired-end (150 bp) sequencing of the three cDNA libraries was performed on the HiSeq 2500 platform (Illumina, USA). To obtain the 5' and 3' terminal sequences of the viral genome, the previously described method was used (Potgieter et al. 2009 (link)). Primers used in this study are listed in Supplementary Table S1 .
10
Profiling lncRNAs from Hair Follicles
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The total RNA was isolated from the hair follicle samples using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The RNA integrity was detected by 1% agarose gel electrophoresis. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) was used to check RNA purity and concentration. First, ribosomal RNA (rRNA) was removed using an EpicentreRibo-Zero™ rRNA Removal Kit (Epicentre, WI, USA). Second, RNA-sequencing libraries were generated under the instructions provided by the NEB Next Ultra™ Directional RNA Library Prep kit for Illumina (New England BioLabs, Inc.) Subsequently, the pooled libraries were sequenced on Hiseq X (Illumina, San Diego, CA, USA), using a chain-specific library construction strategy to count the number and types of lncRNAs.
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