Fragmentation buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

Fragmentation buffer is a solution used to prepare DNA or RNA samples for analysis. Its core function is to physically break down larger nucleic acid molecules into smaller fragments, which can then be analyzed using various techniques such as sequencing or electrophoresis.

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RNA fragmentation buffer (3 citations)

47 protocols using fragmentation buffer

RNA-seq library preparation protocol

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Total RNAs were purified by Trizol LS (Invitrogen 10296028) and
treated with DNase I (Invitrogen Turbo DNase AM2238). mRNAs were isolated
using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs
E7490) and fragmented using Fragmentation Buffer (Ambion AM8740). cDNAs were
synthesized using SuperScript II (Invitrogen 18064014), and non-directional
high-throughput sequencing libraries were prepared using NEBNext Ultra DNA
Library Prep Kit (New England Biolabs, E7370). Libraries were processed on
the Illumina HiSeq platform for single-end 50-nt sequencing. See Quantification and Statistical Analysisfor downstream analyses. RNA-seq experiments are listed in https://github.com/kohta-ikegami/pS22-LMNA/blob/master/datasets.md.

Ikegami K., Secchia S., Almakki O., Lieb J.D, & Moskowitz I.P. (2020). Phosphorylated Lamin A/C in the nuclear interior binds active enhancers associated with abnormal transcription in progeria. Developmental cell, 52(6), 699-713.e11.

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RNA-seq Library Preparation Protocol

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The poly (A) RNA was isolated from 20 μg of the total RNA pool using Dynal oligo (dT) 25 beads (Invitrogen) according to the manufacturer’s protocol. Following purification, the mRNA was fragmented into smaller pieces at 70 °C for 5 min in the fragmentation buffer (Ambion) and reverse-transcribed to synthesize first strand cDNA using SuperScript III reverse transcriptase (Invitrogen) and N6 random hexamers (Takara). Subsequently, second strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase (Invitrogen). These cDNA fragments were further processed by end repair using T4 DNA polymerase, the Klenow fragment of DNA polymerase, and T4 polynucleotide kinase (NEB), and ligation of adaptors with Illumina’s adaptor oligo mix and T4 DNA ligase (Invitrogen). The products were gel purified to obtain DNA approximately 200 bp long using Qiaquick Gel Extraction Kit (Qiagen) and enriched with PCR for preparing the sequencing library. The quality of the cDNA library was examined by Agilent 2100 Bioanalyzer.

Rekik I., Chaabene Z., Grubb C.D., Drira N., Cheour F, & Elleuch A. (2015). In silico characterization and Molecular modeling of double-strand break repair protein MRE11 from Phoenix dactylifera v deglet nour. Theoretical Biology & Medical Modelling, 12, 23.

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Radish Leaf Transcriptome Sequencing

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Total RNA was isolated using Trizol reagents (Invitrogen, USA) according to the manufacturer’s protocol. For cDNA library construction, a total of 20 μg RNA from radish leaf samples at two different stages were pooled in equimolar quantity. Briefly, the mRNA was isolated using magnetic beads with Oligo (dT), and fragmented to small pieces using fragmentation buffer (Ambion, USA). Then the mRNA fragments were used as templates to synthesize double-stranded cDNA with random hexamer primers using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, USA). The synthesized cDNA were purified with QiaQuick PCR extraction kit and subjected to end reparation and single nucleotide A (adenine) addition. Thereafter, the short fragments were connected with adapters and the suitable fragments were screened as templates for PCR amplification. The transcriptome library was sequenced using Illumina HiSeq™ 2000 at Beijing Genomics Institute (Shenzhen, China).

Nie S., Li C., Xu L., Wang Y., Huang D., Muleke E.M., Sun X., Xie Y, & Liu L. (2016). De novo transcriptome analysis in radish (Raphanus sativus L.) and identification of critical genes involved in bolting and flowering. BMC Genomics, 17, 389.

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RNA-seq of Embryonic Development

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At specific developmental stages, whole bodies of embryos or anesthetized larvae were collected and immediately placed in liquid nitrogen until RNA extraction. Total RNA was extracted from 3 specimens per developmental stage using Trizol (Invitrogen, CA, USA) according to the manufacturer’s instructions. The quantity and purity of the extracted RNA were analyzed using Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >8.0. RNA extracted from specimens of each development stage were pooled together as one stage-specific sample. Approximately 10 μg of total RNA was employed for Poly (A) mRNA isolation using oligo-dT magnetic beads (Invitrogen). Subsequently, the mRNA was fragmented into small pieces in the presence of divalent cations (fragmentation buffer (Ambion, #AM8740)) at 94°C for 5 min using an ultrasonicator. The RNA fragments were reverse-transcribed into the cDNA library using the mRNA-Seq preparation kit (Illumina, San Diego, USA). The paired-end sequencing (2*100 bp) on an Illumina Hiseq2000 platform was implemented using paired-end libraries with normal insert size of 300±50 bp.

Song W., Jiang K., Zhang F., Lin Y, & Ma L. (2015). Transcriptome Sequencing, De Novo Assembly and Differential Gene Expression Analysis of the Early Development of Acipenser baeri. PLoS ONE, 10(9), e0137450.

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Transcriptome Profiling of Salt-Tolerant Melilotus

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mRNA was extracted from Melilotus albus and Melilotus siculus using a Qiagen Oligotex mRNA mini kit. Fragmentation of mRNA was done using an Ambion fragmentation buffer. Construction of the cDNA library was based on the Illumina protocol. First strand cDNA synthesis was done using Random Hexamer Primers (Invitrogen) and second strand synthesized using a DNA Polymerase 1 (Promega). End repair was carried out to create uniform blunt ends (Epicentre End-IT repair kit). Unique 4 bp adaptors (Illumina) were added so that the libraries could be pooled for sequencing. An ‘A’ base was added using a Klenow enzyme (3 ^′ to 5 ^′ exo minus, NEB) and adaptor ligation was performed using Epicentre Fast-Link DNA ligation kit. The cDNA template was run on a 2% agarose gel at 120 V for 60 minutes and fragments of approximately 200–500 bp were removed and purified (Zymo gel purification kit). The purified cDNA template was PCR enriched using the Illumina primers and a Phusion polymerase (NEB). The library was quantified using an Invitrogen Qubit fluorometer. Libraries were sequenced on an Illumina Genome Analyzer II under normal conditions and conditions associated with salt tolerance and/or waterlogging tolerance as single-end 100 bp reads, which were trimmed to 71 bp.

Fu S., Chang P.L., Friesen M.L., Teakle N.L., Tarone A.M, & Sze S.H. (2019). Identifying similar transcripts in a related organism from de Bruijn graphs of RNA-Seq data, with applications to the study of salt and waterlogging tolerance in Melilotus. BMC Genomics, 20(Suppl 5), 425.

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Drought-Induced Transcriptome Profiling

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Total RNA was extracted from leaves of five plants that had been subjected to 10-day-drought or control treatments using Promega Total RNA Isolation System (Z3100). Then, mRNA was fragmented in the fragmentation buffer (Ambion). The first strand of cDNA was reversed transcribed with SuperScript II (Invitrogen) using random primer (TAKARA). Next, dTTP was replaced by dUTP for the synthesis of the second strand. The repaired double-stranded cDNA was ligated with Illumina TruSeq adaptor and digested using USER enzyme (NEB). Finally, 350–450 bp fragments were recovered and purified, and 18 cDNA libraries (3 accessions × 2 treatments × 3 replicates) were sequenced on the Illumina HiSeq 2500 platform.

Xu Y.C., Zhang J., Zhang D.Y., Nan Y.H., Ge S, & Guo Y.L. (2021). Identification of long noncoding natural antisense transcripts (lncNATs) correlated with drought stress response in wild rice (Oryza nivara). BMC Genomics, 22, 424.

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Strand-specific RNA-seq Library Preparation

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Strand-specific RNA-seq libraries were prepared in accordance with the protocol described by Waldbauer and coworkers (52 (link)). Briefly, The DNase-treated RNA (100 ng) was fragmented by divalent-cation hydrolysis (Fragmentation Buffer; Ambion, Austin, TX) at 70°C for 12 min to yield fragment sizes between 50 and 300 nt. After precipitation with ethanol, fragments were subjected to poly(A) tailing and end repairing (NEB Reagents). RNA was treated with Antarctic phosphatase (New England Biolabs) and then phosphorylated at the 5′ RNA end with T4 polynucleotide kinase. The transcribing strand was labeled by ligation of a 5′ hybrid DNA-RNA primer and after purification with RNA clean XP beads (Beckman Coulter Genomics), the first-strand cDNA synthesis reaction was carried out with SuperScript II reverse transcriptase (Invitrogen) and Illumina’s poly(T) primer and deoxynucleoside triphosphates (20 mM). Reaction components were removed with Agencourt AMPure XP SPRI beads (Beckman), and primary transcripts were enriched with Pfu Hi Fidelity polymerase (Invitrogen) and the Illumina spacers as primers. Illumina adaptors and bar codes were ligated by PCR in accordance with the manufacturer’s instructions. Libraries were purified with SPRI beads (Beckman) and quantified with a NanoDrop (Bio-Rad). Sequencing was performed with the Illumina technology at Ambry Genetics.

Rodríguez J.G., Hernández A.C., Helguera-Repetto C., Aguilar Ayala D., Guadarrama-Medina R., Anzóla J.M., Bustos J.R., Zambrano M.M., González-y-Merchand J., García M.J, & Del Portillo P. (2014). Global Adaptation to a Lipid Environment Triggers the Dormancy-Related Phenotype of Mycobacterium tuberculosis. mBio, 5(3), e01125-14.

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mRNA Sequencing and Differential Expression

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A minimum of 3 μg of total RNA was oligo (dT) selected using the Dynabeads mRNA purification kit (Invitrogen). The mRNA isolated from total RNA was fragmented into short fragments with a fragmentation buffer (Ambion). Double-stranded cDNA was synthesized with these short fragments as templates. The cDNA was end-repaired, ligated to Illumina adapters, size selected on agarose gel (approximately 250 bp) and PCR amplified. The cDNA library was sequenced on an Illumina HiSeq 2000 sequencing platform (Berry Genomics). The gene expression levels for each transcript were estimated as the number of reads per kilo-base of exon model per million mapped reads (RPKM) using only uniquely mapped reads in exonic regions. A gene is considered significantly differentially expressed if its expression differs between samples from the two groups, NC and miR-3935 mimics, with the log Fold Change > 1 or < -1 and the p value < 0.05 as calculated by Cufflinks.

Jin M., Xu S., Li J., Yao Y, & Tang C. (2021). MicroRNA-3935 promotes human trophoblast cell epithelial-mesenchymal transition through tumor necrosis factor receptor-associated factor 6/regulator of G protein signaling 2 axis. Reproductive Biology and Endocrinology : RB&E, 19, 134.

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Enrichment and Sequencing of CircRNAs

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Ribosome RNA and linear RNA were removed to retain circRNAs. We used fragmentation buffer (Ambion, Foster City, CA, USA) to fragment the enriched circRNA and reverse transcribed the circRNA into cDNA using random primers. The second strand of cDNA was synthesized by DNA polymerase I, RNase H, dNTPs and buffer. The double-stranded cDNA was purified with the QiaQuick PCR extraction kit (Qiagen, USA), end repaired, polyadenylated, and ligated to Illumina sequencing adapters. Then, we used Uracil-N-Glycosylase (UNG) to digest the second-strand cDNA. The fragments were purified by VAHTSTM DNA Clean Beads and enriched by PCR amplification. Finally, the library products were sequenced via Illumina HiSeqTM2500.

Cao H., Liu J., Du T., Liu Y., Zhang X., Guo Y., Wang J., Zhou X., Li X., Yang G, & Shi X. (2021). Circular RNA screening identifies circMYLK4 as a regulator of fast/slow myofibers in porcine skeletal muscles. Molecular Genetics and Genomics, 297(1), 87-99.

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RNA-seq Analysis of Transcriptome Profiling

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Samples were processed and analyzed as previously described^{12 (link)} with minor modifications as indicated below:
RNA was extracted from Trizol-lysed cells and 1 μg of total RNA was used for each sample. Polyadenylated RNA was selected using Ambion Dynabeads mRNA Purification Kit (Life Technologies 61006) and fragmented with Fragmentation Buffer (Ambion, #AM8740). First strand synthesis was performed using Random Hexamer Primers (Invitrogen, #48190-011) and SuperScript II (Invitrogen, #18064-014). Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014).
Libraries were end-repaired, 3′ A-tailed, and ligated to NEBNext Multiplex Oligo Adaptors (NEB E7335S). Sequencing was performed on an Illumina NextSeq 500 by Stanford Functional Genomics Facility.
Reads were mapped to hg19 annotation using Tophat2^{38 (link)} (version 2.0.13) and transcript expression was quantified against RefSeq gene annotations using featureCounts^{39 (link)}. Differential testing and log2 fold change calculation was performed using DESeq2^{40 (link)} with default multiple hypothesis adjustment to reduce false positives (Benjamini-Hochberg, FDR = 0.1). Gene Ontology analyses were performed using DAVID^{41 (link),42 (link)}.

Venkatesh H.S., Tam L.T., Woo P.J., Lennon J., Nagaraja S., Gillespie S.M., Ni J., Duveau D.Y., Morris P.J., Zhao J.J., Thomas C.J, & Monje M. (2017). Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma. Nature, 549(7673), 533-537.

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