For each library, around 1 μg of purified PCR product were fragmented by nebulization (2.1 bars of nitrogen for 1 min) and subjected to library preparation with the Rapid Library Preparation Kit (Roche) according to manufacturer’s instructions. The different samples were labeled via ligation of Multiplex Identifiers (MID) oligonucleotide adaptors to allow multiplexing. Three MID-containing libraries were quantified using the Qubit® Fluorometer and pooled in equimolar amounts into a single sample prior to emulsion PCR amplification and sequencing in parallel on the Roche GS Junior 454 sequencer giving a mean total number of 69,060 reads (387 bp mean length) per library. The data obtained were then sorted according to their tag sequences.
Rapid library preparation kit
Manufactured by Roche
Sourced in Switzerland
The Rapid Library Preparation Kit is a lab equipment product designed for the preparation of DNA libraries. It provides a streamlined process for library construction, enabling efficient and quick sample preparation for downstream analysis.
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Lab products found in correlation
Gs junior 454 sequencer, by Roche (1 mentions)
454 gs flx sequencer, by Roche (1 mentions)
Ampure beads, by Agilent Technologies (1 mentions)
Tbs 380 fluorometer, by Promega (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Faststart high fidelity pcr system, by Roche (1 mentions)
Illustra gfxtm pcr dna and gel band purification kit, by GE Healthcare (1 mentions)
Peltier thermal cycler, by Bio-Rad (1 mentions)
Gs junior system, by Roche (1 mentions)
Hiseq 1000, by Illumina (1 mentions)
Trueseq dna sample preparation kit, by Illumina (1 mentions)
Gotaq long pcr master mix, by Promega (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Casava 1, by Illumina (1 mentions)
454 flx titanium, by Roche (1 mentions)
Gs flx titanium general library preparation kit, by Roche (1 mentions)
Gs junior titanium sequencing kit, by Roche (1 mentions)
454 gs flx platform, by Roche (1 mentions)
Gs reference mapper, by Roche (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
9 protocols using rapid library preparation kit
1
Roche 454 Sequencing Library Preparation
2
RNA-seq Analysis of Coral Symbiosis
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mRNA from cured and symbiotic samples were fragmented and processed following the manufacturer’s protocol (Roche). AMPure beads (Agilent) were used to purify the newly synthesized cDNAs, followed by cDNA-ends repair and RL adaptor ligation (Rapid Library Preparation kit, Roche). Afterwards, residual small cDNA fragments were removed using AMPure beads (Agilent). Both cured and symbiotic white body libraries were quantified against the RL standard (RL cDNA synthesis kit, Roche) using a TBS 380 Fluorometer (Turner Biosystems). Quality and size of the cDNA libraries were verified using High Sensitivity DNA chips on an Agilent 2100 BioAnalyzer. Sequencing was completed using a Roche 454 GS FLX+ sequencer (Genomics Lab, NMSU), and each sample was run on two separate quarters of a plate. Sequences can be accessed on the NCBI SRA (accession number SRP049997).
3
nifH Gene Amplification and Sequencing
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Genomic DNA samples were used as templates for amplification of the nifH gene of approximately 360 bp following the nested PCR protocols of Zehr et al. (1998) (link) and using a FastStart High Fidelity PCR system, dNTPack (Roche, Switzerland) with a Peltier Thermal Cycler (Bio-Rad, USA). In order to enable sample multiplexing during sequencing, barcodes were incorporated between the adapter and forward primer. Nuclease-free water was used as the negative control in each reaction. Triplicate PCRs were performed for each sample and the amplicons were pooled and subsequently purified with the illustraTMGFXTMPCR DNA and Gel Band Purification kit (GE Healthcare, Little Chalfont, Bucks, UK). An amplicon library was constructed with equimolar concentrations of the amplicons, and emPCR was conducted according to the Rapid Library preparation kit instructions (Roche, Switzerland). DNA beads were successfully deposited onto the PicoTiterPlate and sequenced with a GS Junior system (Roche, Switzerland).
4
Whole Genome Shotgun Sequencing Protocols
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Whole genome shotgun DNA library was prepared using Illumina TrueSeq DNA sample preparation kit (FC-121-2001). The paired-end (PE) (2 × 100 nts) sequencing was carried out using Illumina HiSeq 1000 at the Next Generation Genomics Facility at Centre for Cellular and Molecular Platforms (C-CAMP). We prepared whole genome shotgun 454 library for Genotype 1 (GKVK, Bangalore, India) using the rapid library preparation kit from Roche (Cat. No. 05608228001y; version 4.0.12). The 454 sequencing was carried out using GS FLX+ chemistry as per Roche/454 manual instructions (http://454.com ).
5
Long-Range PCR for DMD Transcript
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DMD-targeted RNA-seq was performed as previously described [15 (link)]. Briefly, the full-length DMD cDNA was amplified as a single long fragment (11.3 kb) by using the GoTaq® Long PCR Master Mix (Promega, Charbonnières-les-Bains, France) and primers located in exon 1 and in the 3′ UTR of the muscle isoform (Dp427m). The library was prepared from 700 ng of purified LR-PCR products using the Rapid Library Preparation Kit (Roche, Basel, Switzerland). Three Multiplex Identifiers (MID)-tagged samples were pooled for simultaneous amplification and sequencing on the Roche GS Junior 454 sequencer (Roche, Basel, Switzerland).
6
High-Quality Nuclear DNA Extraction from Plants
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The plant material was provided by the Genebank of the National Institute of Agrobiological Sciences (NIAS). High-quality nuclear DNA with reduced organellar DNA was extracted from young leaves using a protocol designed for BAC DNA extraction with some modifications [10 ]. All sequencing reads were obtained using the Illumina HiSeq2000 at Operon Biotechnologies, Inc. (Eurofins Genomics). Standard short-read libraries and mate-paired libraries with 8 kbp insertion were built using the TruSeq SBS v5 for sequencing runs at 2 × 100 bp or 200 bp total. After sequencing, HiSeq Control Software v.1.4.8 and CASAVA 1.8.1 (Illumina) were utilized for base calling. Single-ended libraries and 3 kbp pair-ended libraries constructed with the GS FLX Titanium General Library Preparation Kit and Rapid Library Preparation Kit (Roche) were sequenced on Roche 454 FLX Titanium at the NIAS, and base calling was performed using the 454 FLX Titanium base caller.
7
FV3 Genome Sequencing Protocol
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Standard kits and protocols developed by the manufacturer were used to sequence the SSME sample on a 454 GS-FLX platform (Roche Diagnostics Corporation). Briefly, a Rapid Library Preparation Kit (Roche, Mississauga, ON) was used to mechanically shear 500 ng of template DNA into short fragments. A universal sequencing primer that included a short DNA sequence unique to the sample (MID tag) was then annealed to both ends of each DNA fragment. A GS Junior Titanium Emulsion PCR Kit (Roche, Mississauga, ON) was used to amplify the sample library, which was sequenced using a GS Junior Titanium Sequencing Kit (Roche, Mississauga, ON). In order to assemble a full genomic sequence, the short sequences produced by 454 sequencing were aligned with the reference FV3 genome, wt-FV3, using GS Reference Mapper (Roche, Mississauga, ON). Any gaps in the assembled genome were then sequenced using custom PCR primers specific to each gap, with sequencing performed by the Robarts Sequencing Facility (London, ON). The final genomic sequence was deposited in GenBank accession number KJ175144.
8
16S Metagenomic Sequencing of Gut Microbiome
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DNA was extracted from faecal material using a QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany) and 50 ng were used for cDNA library synthesis with the Rapid Library Preparation Kit (Roche Applied Science, Mannheim, Germany), according to the manufacturer’s instructions. The cDNA was analysed with a Bioanalyzer and High Sensitive DNA Kit (Agilent Technologies Inc., Santa Clara, CA, United States) to ensure equimolar use of the samples in PCR. These samples were then sequenced with a 16S Metagenomic Sequencing® Illumina Kit combined with the HiSeq 2500 System (Illumina) sequencer, according to the manufacturer’s instructions. Sequence reads obtained from the V4 region of the 16S gene were analysed according to the UPARSE pipeline[24 (link)], using the USEARCH v9.2.64 package. For OTU clustering a threshold of 97% similarity was used through the UPARSE-OTU algorithm. α- and β-diversity analyses were calculated using the R package Phyloseq v.1.19.1[25 (link)] and vegan 2.4_2 packets, using the OTU table normalized to the smallest sample size. Taxa with differential abundance between groups were identified using the Kruskal-Wallis test (P < 0.05). In the bar plot are shown those taxa with relative abundance greater than 1%.
9
Targeted Sequencing of ABCC8 and ABCD2
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Libraries encompassing all exons of ABCC8 (39 exons) and ABCD2 (10 exons) genes were prepared according to the manufacturer (Roche, Prague, Czech Republic). Based on the character of probe design, i.e. tiling; the exons were surrounded by approximately 30 bp regions of intronic sequences which were also sequenced in both directions. Target enrichment was performed using SeqCap EZ Choice by Nimblegen. Libraries were prepared using Rapid Library Preparation Kit (Roche). Samples were sequenced on 454 GS Junior system (Roche).
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