The library for small RNA-sequencing was prepared according to the NEBNext Multiplex Small RNA Library Prep Set for Illumina manual from New England Biolabs (NEB, Herts, UK). The main steps for the preparation of small RNA libraries using this kit were as follows: (1) 3′ ligation, (2) primer hybridization, (3) 5′ ligation, (4) first-strand synthesis, (5) PCR amplification, and (6) size selection. The molarity and size of the libraries were assessed by an Agilent high-sensitivity chip on a 2100 Bioanalyzer (Agilent Technologies, Germany). An individual library was prepared for each sample. Sequencing of 51 bp single-end reads was performed on the Illumina MiSeq at the Laboratory of Immunotherapeutic and Vaccine, Universiti Putra Malaysia.
Agilent high sensitivity chip
Manufactured by Agilent Technologies
Sourced in Germany
The Agilent high-sensitivity chip is a laboratory equipment designed for analytical applications. It provides high-sensitivity detection capabilities for various analytes. The core function of this product is to enable precise and accurate measurements in a range of scientific research and testing scenarios.
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Lab products found in correlation
Ion xpress barcode adapter, by Thermo Fisher Scientific (3 mentions)
Ion proton sequencer, by Thermo Fisher Scientific (3 mentions)
Ion rna seq kit v2, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Quant it dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
E gel ex, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Truseq sbs kit v3, by Illumina (1 mentions)
Rnase free dnase 1, by Promega (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (1 mentions)
6 protocols using agilent high sensitivity chip
1
Small RNA Sequencing Library Preparation
2
Paired-End Sequencing of Human DNA
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Completed libraries were pooled for two rounds of size selection using a 2% and then 1% agarose gel (E-Gel Ex, Invitrogen) to excise the 200- to 400-bp range. Library size distribution was confirmed using an Agilent High Sensitivity chip (Agilent), and the final concentration was determined using a Quant-iT dsDNA HS assay kit and Qubit fluorometer (Invitrogen). Libraries were submitted for sequencing to the Michael Smith Genome Sciences Centre (Vancouver, Canada), where paired-end 100-nt reads were generated on the HiSeq 2000 (SBSxx) platform. For read metrics of each individual library, see Supplemental Table S1 . The .fastq files were aligned to the human reference assembly (hg19/GRCh37, released Feb 2009) and analyzed using the open source software ‘Bioinformatic Analysis of Inherited Templates’ (BAIT) (Hills et al. 2013 (link)).
3
Transcriptome Analysis of Fungal Challenged CoC 671
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Total RNA was extracted from CoC 671 challenged with Cf671 using TRI Reagent (Sigma-Aldrich, USA) in different time intervals 2,4,6 dpi, and treated with Rnase free DNAse I (Promega, USA) Subsequently, the quality of RNA was checked in 1% denatured agarose gel electrophoresis for the presence of intact 28 and 18S bands and RNA was quanti ed using Nanodrop-8000. The paired-end cDNA sequencing library was prepared using Illumina TruSeq SBS Kit v3 as per the manufacture protocol. Library preparation was started with mRNA fragmentation followed by reverse transcription, second-strand synthesis, paired-end adapter ligation and nally ended with index PCR ampli cation of adaptor-ligated library. Library quanti cation and quality check was performed on Caliper Lab Chip GX using HT DNA High Sensitivity Assay Kit. The libraries of all samples were in the size range of 200 bp to 600bp. The resulting libraries were validated using the Agilent Bio Analyzer 2100 onto the Agilent High Sensitivity Chip performed at Nucleome Informatics Pvt. Ltd, Hyderabad, India.
4
Ion RNA-Seq Library Preparation
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Library preparation was performed using the Ion Total RNA-Seq Kit v2 (Life Technologies). Large RNA libraries were prepared using the whole transcriptome protocol provided with the kit, and small RNA libraries were prepared using the small RNA protocol. Large and small RNA libraries were barcoded using Ion Xpress Barcode Adapters (Life Technologies). Quality control was performed using Agilent High Sensitivity chips (Agilent) and Experion DNA 1K kits (Bio-Rad). Libraries were loaded onto Ion PI chips via the Ion PI Template OT2 200 v3 (for small RNAs) or v2 (for large RNAs), and Ion PI Sequencing 200 v3 (for small RNAs) or v2 (for large RNAs) Kits, and sequenced on an Ion Proton Sequencer (Life Technologies).
5
Small RNA-seq Library Preparation
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Prior to library preparation, total sperm RNA samples were enriched for small RNAs using the protocol provided in the Ion RNA-Seq Kit v2 (Life Technologies). Small RNA-enriched samples were used for small RNA library preparation, using the same kit, and barcoded with Ion Xpress Barcode Adapters (Life Technologies). Quality control was performed using Agilent High Sensitivity chips (Agilent). Libraries were loaded onto the same Ion PI chip via the Ion PI Template OT2 200 v3 and Ion PI Sequencing 200 v3 kits, and sequenced on an Ion Proton Sequencer (Life Technologies).
6
Small RNA-seq Library Preparation
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