Small RNA-Seq was performed as described in [54 (link)]. Briefly, 500 ng of total RNAs were ligated, reverse transcribed and amplified (18 cycles) with the reagents from the NextFlex small RNAseq kit V3 (Bioo scientific, Villebon-sur-Yvette, France). Amplified libraries were quantified with the Bioanalyzer High Sensitivity DNA Kit (Agilent, Les Ulis, France), pooled and size-selected from 140 nt to 170 nt with the LabChip XT DNA 300 Assay Kit (Caliper Lifesciences, Villepinte, France). Libraries were then sequenced on an Illumina Nextseq 500 Mid Flowcell with 75 pb reads for a total of 147 M reads.
Nextflex small rna seq kit v3
Manufactured by PSC Biotech
Sourced in United States
The NEXTflex Small RNA-Seq Kit v3 is a library preparation kit designed for sequencing small RNA molecules, such as microRNAs, from a variety of sample types. The kit utilizes a proprietary ligation-based approach to generate sequencing-ready libraries from small RNA inputs.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500, by Illumina (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Fragment analyzer, by Agilent Technologies (3 mentions)
Nextseq 550 sequencer, by Illumina (2 mentions)
Hiseq 2500 instrument, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Novaseq reagent kit, by Illumina (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Miseq system, by Illumina (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
X ten sequencing platform, by Illumina (1 mentions)
Ribominus plant kit for rna seq, by Thermo Fisher Scientific (1 mentions)
28 protocols using nextflex small rna seq kit v3
1
Small RNA Sequencing Workflow
2
Small RNA-Seq library preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10 ng of total RNA was used as input for library preparation with the NEXTflex Small RNA-Seq kit V3 (Bioo Scientific, Austin, TX) following the manufacturer’s instructions with sample amplification for 23 cycles. The libraries were size selected using the Pippin prep per the Nextflex protocol and eluted bands between 115 and 170 bp resulting in a product around 150 bp. (Sage Science, Beverly, MA) and purified together per manufacturer’s protocol. Nine uniquely indexed libraries were pooled in equimolar ratio based on concentration between 145–160 bp as was determined by Agilent Bioanalyzer. The pools were size selected and purified together per manufacturer’s protocol and run on a lane of HiSeq3000 as single read 50 cycles.
3
Small RNA-Seq Protocol for Exosomes and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10 ng (EVs) or 50 ng (cells) of total RNA was used in the small RNA protocol with the NEXTflex Small RNA-seq Kit v3 (Bioo Scientific) according to the instructions of the manufacturer. A pool of libraries was used for sequencing at a concentration of 10 nM. Sequencing of 1x75 bp was performed with an Illumina NextSeq 550 sequencer at the sequencing core facility of the IZKF Leipzig (Faculty of Medicine, University Leipzig) according to the instructions of the manufacturer. Demultiplexing of raw reads, adapter trimming and quality filtering was done according to Stokowy et al. (25 (link)), using the adapter sequences of the NEXTflex kit containing random bases next to the library insert. Mapping against the human reference genome (hg38) and miRbase reference sequences (v22) was done using Bowtie2 (26 (link)). Read counts were calculated with the R bioconductor package Rsamtools (http://bioconductor.org/packages/release/bioc/html/Rsamtools.html ) and normalised using the DESeq2 (27 (link)) and EdgeR (28 (link)) R bioconductor packages.
4
Spinal Cord Cell Sorting and RNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spinal cords of P0/P1 Hlxb9-GFP and Clp1R140H/−; Hlxb9-GFP mice were dissociated and lightly fixed in preparation for cell sorting using a protocol adapted from previous publications (SI Appendix, Supplementary Text ) (41 (link), 42 (link)). RNA samples were pooled so that the input for each library consisted of 175 ng of RNA derived from six spinal cords. Two wild-type and two compound heterozygote 3′READS+ libraries were prepared as described (20 (link), 43 ), with some modifications: RNase III fragmentation was reduced to 5 min and adapter and primer sequences came from the NEXTFLEX Small RNA-Seq Kit v3 (Bioo Scientific); 100-nt paired-end reads were generated on a HiSeq 4000.
5
Small RNA-Seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NGS library prep was performed with NEXTflex Small RNA-Seq Kit V3 following Step A to Step G of Bioo Scientific's standard protocol (V16.06). Libraries were prepared with a starting amount of 1 ng and amplified in 25 PCR cycles. Amplified libraries were purified by running an 8% TBE gel and size-selected for 18-40 nt. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life Technologies). Samples from 1, 3 and 6 dpf and samples for zygote and 10 dpf PGCs were pooled in equimolar ratio and sequenced on 2 NextSeq 500 Flowcell, PE for 2×75 cycles plus 16 cycles for the index read.
6
Profiling Postpartum Maternal miRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yield and quality of the RNA samples was assessed using an Agilent 2100 Bioanalyzer prior to library construction with the NEXTflex Small RNA-Seq Kit v3 (Bioo Scientific; Austin, Texas). Multiplexed samples were run on an Illumina HiSeq 2500 Instrument at a targeted depth of one million reads per sample. FastQ outputs were clipped, trimmed, and filtered to a maximum read length of 30 base pairs using the FASTX Toolkit Module in Mobaxterm. Reads were aligned to the hg38 build of the human genome in Partek Flow (Partek; St. Louis, Missouri) using the Bowtie 2 algorithm. Total miRNA counts within each sample were quantified with miRBase mature microRNAs v21 and read counts were normalized across samples using a trimmed mean of M-values (TMM) method. Only miRNAs with raw read counts greater than five in at least 50 % of samples were evaluated in the differential expression analysis. A multi-model approach employing gene set analysis (GSA) in Partek Flow was used to identify individual miRNAs with differential expression between tMBM and pMBM samples collected 3–4 weeks post-delivery (in lipid and skim fractions respectively). Differential expression was defined as a Benjamini Hochberg False Discovery Rate (FDR) < 0.05. The data set supporting the results of this article will be available in the NCBI Sequence Read Archive.
7
Small RNA Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this study, 10 to 50 ng of total RNA was used in the small RNA protocol with the NEXTflex Small RNA-seq Kit v3 (Bioo Scientific, Austin, USA), according to the manufacturer’s instructions. A pool of 12 libraries was used for cluster generation at a concentration of 10 nM using an Illumina cBot. Sequencing of 50 bp was performed with an IlluminaHighScan-SQ sequencer at the sequencing core facility of IZKF Leipzig (Faculty of Medicine, University Leipzig) using version 3 chemistry and flow cell according to the manufacturer’s instructions.
8
Transcriptome Profiling of Cellular Perturbation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were plated on six-well plates and grown to 50% density. The cells were transfected with 2 μg/well of U6-MosIR, U6-Lin28aIR, CAG-EGFP-MosIR, or CAG-EGFP-Lin28IR plasmids, cultured for 48 h, washed with PBS, and total RNA was isolated using RNAzol (MRC) according to the manufacturer's protocol. RNA quality was verified by Agilent 2100 Bioanalyzer. The library construction and high-throughput sequencing of the RNA transcriptome were performed either from small RNA (<200 nt) fraction using SOLiD (version 4.0) sequencing platform (Seqomics) or libraries were constructed from total RNA using NEXTflex Small RNA-Seq Kit v3 (Bioo Scientific) according to the manufacturer's protocol and sequenced on the Illumina HiSeq2000 platform at the Genomics Core Facility at EMBL. High-throughput sequencing data were deposited in the GEO database (GSE41207 for SOLiD data and GSE126324 for Illumina data).
9
Plasma and Liver Small RNA Sequencing in NAFLD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small RNA seq (< 50 nt) was performed on pooled plasma and liver tissue from 5 patients with histopathologically confirmed NAFLD and 5 patients without NAFLD. Total RNA was extracted from liver or plasma using TRIzol LS Reagent (Invitrogen, USA). The small RNA sequencing library was prepared with a NEXTflex Small RNA-Seq Kit v3 (BIOO SCIENTIFIC, USA) following the manufacturer’s protocol and sequenced on an Illumina X Ten sequencing platform (Aksomics, China).
10
Ribosome Footprint Profiling of Arabidopsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total ribosomal footprints were obtained from a mix of flower buds and open flowers of Col-0 and dweorg1 plants grown in the greenhouse, as described in Planchard et al.25 (link). The total RNA and ribosome footprints were depleted of rRNAs using the RiboMinus™ Plant Kit for RNA-Seq (invitrogen), following the manufacturer’s instructions. The sequencing libraries of total RNA and ribosomal footprints of two biological repeats were prepared using the NEXTflex Rapid Directional RNA-Seq Kit (Bioo Scientific) and the NEXTflex Small RNA-Seq Kit v3 (Bioo Scientific), respectively. Next-generation sequencing was performed by the sequencing facility of the Institut de Biologie Intégrative de la Cellule (Gif-sur-Yvette, France) using Illumina NextSeq technology (single end, 75 nt). Bioinformatic analysis was performed as described in Planchard et al.25 (link).
All experimental research did fully comply with the relevant institutional, national, and international guidelines and legislation.
All experimental research did fully comply with the relevant institutional, national, and international guidelines and legislation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!