Small RNA-seq libraries were constructed from fecal RNA isolates using NEXTflex™ Small RNA-Seq Kit (Bioo Scientific Corporation., USA). 500 ng of RNA was used as input material. The library was prepared with a unique indexed primer so that libraries could be pooled into one sequencing flow cell. Multiplex adaptor ligations, primer hybridization, reverse transcription reaction and PCR amplification were performed according to the manufacturer’s protocol. Libraries were further purified with a gel size selection using Blue Pippin (Sage Science, Inc. USA). The obtained libraries were checked for quality with Agilent 2200 TapeStation and were sequenced with the Illumina NextSeq 500 System (50 nt, single read) at the Biopolymers Facility at Harvard Medical School. Data were analyzed following the exceRpt small RNA-seq pipeline V4.6.2 (Subramanian et al. 2015 (link)). Normalization and differential expression were performed with the R package DESeq2 v.1.10.1 (R version 3.3.2) (Love et al. 2014 (link)). Sequencing data are available at Sequence Read Archive (SRA)(https://www.ncbi.nlm.nih.gov/sra ).
Nextflex small rna seq kit
Manufactured by PSC Biotech
Sourced in United States
The NEXTflex Small RNA-Seq Kit is a laboratory tool designed for the preparation and sequencing of small RNA samples. It provides a standardized workflow for the isolation, library construction, and subsequent analysis of small RNA molecules such as microRNAs, siRNAs, and other noncoding RNAs.
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Lab products found in correlation
Nextseq 500 system, by Illumina (1 mentions)
Bluepippin, by Sage Science (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
2100 bioanalyser, by Agilent Technologies (1 mentions)
Dynabeads mrna direct kit, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Bcl2fastq 2.20, by Illumina (1 mentions)
Sequencing platform, by Illumina (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using nextflex small rna seq kit
1
Small RNA-seq of Fecal Samples
2
Profiling Small RNA Expression in A. baumannii
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Total RNA was extracted from A. baumannii AYE cells expressing the WCHA45 creTA (ΨR2-replaced) as described above. A total of 50 μg of RNA was treated with T4 polynucleotide kinase (New England Biolabs, MA, USA) according to the manufacturer’s protocol at 37 °C. And then the kinase was inactivated by incubating at 65 °C for 20 min. The treated RNA was purified using the phenol-chloroform method, precipitated with an equal volume of isopropanol and 0.1 volume of 3 M sodium acetate. The RNA sample was re-dissolved in RNA-free water for sequencing. A small RNA library was constructed for the RNA molecules ranging from 30 to 300 nt with the NEXTFLEX Small RNA-Seq Kit (Bioo Scientific, TX, USA), and then subjected to Illumina HiSeq sequencing (paired-end, 150 bp reads). The raw data reads were processed to remove adapters and mapped to the creA sequence using previously reported Perl scripts19 (link).
3
Transcriptome Analysis via RNAseq and miRseq
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PolyA mRNA was isolated from total RNA using the Dynabeads mRNA DIRECT Kit (Life Technologies, UK) and verified using a Bioanalyser-2100 (Agilent, Santa Clara, CA, USA). RNAseq was carried out essentially as described in Dart et al., 2017 [16 (link)]. For MiR-seq we utilised NEXTFlex Small RNA-seq kit (Bioo Scientific, Austin, TX, USA).
4
Small RNA Sequencing of Bovine and Human Homologs
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The NEXTflex Small RNA-Seq Kit (Bioo Scientific) was used to prepare small RNA libraries from each matrix, which were then sequenced on an Illumina NovaSeq 6000 utilizing a 50-base single-end sequencing method by CeGaT GmbH (Tübingen). Illumina bcl2fastq (2.20) was used to demultiplex the sequencing reads, and Skewer was used to trim the adapters (version 0.2.2). Demultiplexed reads were analysed using sRNAbench [1 (link)] and reads with an average PHRED score below 20 were discarded. To identify bovine (bta) miRNAs and human (hsa) miRNA homologues, the analysis was run in genome mode with the cow genome (UMD3 1 mp) and miRBase 22 as a reference. Reads below 15 nucleotides were discarded before mapping and were not included in further analyses. Bowtie was used to map sequence reads against the miRNA library allowing two nucleotide mismatches. Data on raw sequencing can be found in the GEO database (GSE198854).
5
Small RNA-Seq Library Preparation
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The libraries were prepared for 50 bp single-end sequencing using the NEXTflex Small RNA-Seq Kit (Bioo Scientific Corp). Namely, small RNA molecules were isolated from 1 μg of total RNA via the adapter ligation. The isolated small RNAs were synthesized as single-stranded cDNAs through the RT (Reverse transcription) priming. By applying this as a template for second-strand synthesis, double-stranded cDNA was prepared through PCR (Polymerase Chain Reaction). And, the fragments around 150 bp were extracted for sequencing through size selection by gel electrophoresis. The quality of these cDNA libraries was evaluated with the Agilent 2100 BioAnalyzer (Agilent, CA, USA) followed by quantification with the KAPA library quantification kit (Kapa Biosystems, MA, USA) according to the manufacturer’s protocol. Following cluster amplification of denatured templates, sequencing was progressed as single-end (50 bp) using Illumina sequencing platform (Illumina, CA, USA).
6
Transcriptome Analysis of ADAR Knockdown
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