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Truseq dna sample kit

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The TruSeq DNA Sample kit is a library preparation kit designed for DNA sequencing. It is used to prepare DNA samples for downstream sequencing applications on Illumina sequencing platforms.

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Lab products found in correlation

Tapestation 2200, by Agilent Technologies (5 mentions) S2 system, by Covaris (5 mentions) Nugen ovation v2 kit, by Tecan (4 mentions) Bcl2fastq2 conversion software v2, by Illumina (4 mentions) Rneasy mini kit, by Qiagen (4 mentions) Nextseq 500, by Illumina (4 mentions) 2100 bioanalyzer, by Agilent Technologies (2 mentions) Hiseq 2000, by Illumina (2 mentions) 2100 expert software, by Agilent Technologies (2 mentions) Cebpa, by Santa Cruz Biotechnology (1 mentions) Rabbit igg, by Merck Group (1 mentions) Genome analyzer, by Illumina (1 mentions) Ovation v2 kit, by Tecan (1 mentions)

7 protocols using truseq dna sample kit

1

Microglial RNA Sequencing Protocol

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RNA clean-up from isolated microglial cells (50,000), TRAP samples and 5% of the unbound fractions from TRAP samples was performed using RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. RNA integrity was assayed using an RNA Pico chip on a Bioanalyzer 2100 (Agilent, Santa Clara, CA), and only samples with RIN>9 were considered for subsequent analysis. Double-stranded cDNA was generated from 1-5 ng of RNA using Nugen Ovation V2 kit (NuGEN, San Carlos, CA) following manufacturer’s instructions. Fragments of 200 bp were obtained by sonicating 500 ng of cDNA per sample using the Covaris-S2 system (Duty cycle: 10%, Intensity: 5.0, Bursts per second: 200, Duration: 120 seconds, Mode: Frequency sweeping, Power: 23W, Temperature: 5.5°C - 6°C, Covaris Inc., Woburn, MA). Subsequently, these fragments were used to produce libraries for sequencing by TruSeq DNA Sample kit (Ilumina, San Diego, CA, USA) following manufacturer’s instructions. The quality of the libraries was assessed by 2200 TapeStation (Agilent). Multiplexed libraries were directly loaded on NextSeq 500 (Ilumina) with High Output single read sequencing for 75 cycles. Raw sequencing data was processed using Illumina bcl2fastq2 Conversion Software v2.17.
Ayata P., Badimon A., Strasburger H.J., Duff M.K., Montgomery S.E., Loh Y.H., Ebert A., Pimenova A.A., Ramirez B.R., Chan A.T., Sullivan J.M., Purushothaman I., Scarpa J.R., Goate A.M., Busslinger M., Shen L., Losic B, & Schaefer A. (2018). Epigenetic regulation of brain region-specific microglia clearance activity. Nature neuroscience, 21(8), 1049-1060.
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2

Microglial RNA Sequencing Protocol

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RNA clean-up from isolated microglial cells (50,000), TRAP samples and 5% of the unbound fractions from TRAP samples was performed using RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. RNA integrity was assayed using an RNA Pico chip on a Bioanalyzer 2100 (Agilent, Santa Clara, CA), and only samples with RIN>9 were considered for subsequent analysis. Double-stranded cDNA was generated from 1-5 ng of RNA using Nugen Ovation V2 kit (NuGEN, San Carlos, CA) following manufacturer’s instructions. Fragments of 200 bp were obtained by sonicating 500 ng of cDNA per sample using the Covaris-S2 system (Duty cycle: 10%, Intensity: 5.0, Bursts per second: 200, Duration: 120 seconds, Mode: Frequency sweeping, Power: 23W, Temperature: 5.5°C - 6°C, Covaris Inc., Woburn, MA). Subsequently, these fragments were used to produce libraries for sequencing by TruSeq DNA Sample kit (Ilumina, San Diego, CA, USA) following manufacturer’s instructions. The quality of the libraries was assessed by 2200 TapeStation (Agilent). Multiplexed libraries were directly loaded on NextSeq 500 (Ilumina) with High Output single read sequencing for 75 cycles. Raw sequencing data was processed using Illumina bcl2fastq2 Conversion Software v2.17.
Ayata P., Badimon A., Strasburger H.J., Duff M.K., Montgomery S.E., Loh Y.H., Ebert A., Pimenova A.A., Ramirez B.R., Chan A.T., Sullivan J.M., Purushothaman I., Scarpa J.R., Goate A.M., Busslinger M., Shen L., Losic B, & Schaefer A. (2018). Epigenetic regulation of brain region-specific microglia clearance activity. Nature neuroscience, 21(8), 1049-1060.
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3

ChIP-Seq Analysis of Transcription Factors

Cited 2 times
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ChIP was performed as previously described29 (link) using the following antibodies: PU.1 (Santa Cruz Biotechnologies, sc352x), CEBPA (Santa Cruz Biotechnologies, sc61x), H3AcK27 (Abcam, Cambridge, UK; ab4729) and rabbit IgG (Sigma, I5006). Library construction was performed using the Illumina TruSeq DNA Sample Kit (Illumina, Cambridge, UK) according to the manufacturer's instructions. Sequencing was performed on the Illumina HiSeq 2000 platform. Reads were mapped to the mm10 mouse reference genome using Bowtie2.30 (link) Mapped reads were converted to density plots and displayed as UCSC genome browser custom tracks, and peaks called using MACS2.31 (link)Using BEDTools,32 (link) ChIP-Seq peak coordinates were combined between PU.1− and PU.1+ conditions for each TF ChIP, and peaks overlapping by at least 1 bp were merged. Coverage scores were counted using the intersectBed function for each merged peak region, and then normalised for peak length and total read counts (per 1 million reads). For H3K27Ac, read coverage regions were extended to 800 bp, and then normalised as above.
Sive J.I., Basilico S., Hannah R., Kinston S.J., Calero-Nieto F.J, & Göttgens B. (2015). Genome-scale definition of the transcriptional programme associated with compromised PU.1 activity in acute myeloid leukaemia. Leukemia, 30(1), 14-23.
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4

RNA-Seq Analysis of TRAP Samples

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RNA purification from TRAP samples and from 5% of their corresponding unbound fractions was performed using RNeasy Mini Kit (Qiagen) following manufacturer’s instructions and used for subsequent sequencing. RNA integrity was assayed by using an RNA Pico chip on Bioanalyzer 2100 using 2100 Expert Software (Agilent, Santa Clara, CA) and only samples with RIN>9 were considered for subsequent analysis. Double-stranded cDNA was generated from 1-5 ng RNA using Nugen Ovation V2 kit (NuGEN, San Carlos, CA) following manufacturer’s instructions. 500 ng of cDNA per sample was sonicated to obtain fragments of 200 base pairs using Covaris-S2 system (Duty cycle: 10%, Intensity: 5.0, Bursts per second: 200, Duration: 120 seconds, Mode: Frequency sweeping, Power: 23W, Temperature: 5.5°C - 6°C, Covaris Inc., Woburn, MA). These fragments were used to produce libraries for sequencing by TruSeq DNA Sample kit (Ilumina, San Diego, CA, USA) following manufacturer’s instructions. The quality of the libraries was assessed by the 2200 TapeStation (Agilent). Multiplexed libraries were directly loaded on NextSeq 500 (Ilumina) with High Output single read sequencing using 75 cycles. Raw sequencing data was processed with Illumina bcl2fastq2 Conversion Software v2.17.
Badimon A., Strasburger H.J., Ayata P., Chen X., Nair A., Ikegami A., Hwang P., Chan A.T., Graves S.M., Uweru J.O., Ledderose C., Kutlu M.G., Wheeler M.A., Kahan A., Ishikawa M., Wang Y.C., Loh Y.H., Jiang J.X., Surmeier D.J., Robson S.C., Junger W.G., Sebra R., Calipari E.S., Kenny P.J., Eyo U.B., Colonna M., Quintana F.J., Wake H., Gradinaru V, & Schaefer A. (2020). Negative feedback control of neuronal activity by microglia. Nature, 586(7829), 417-423.
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5

Parachlamydia Bn9 DNA Sequencing Protocol

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Parachlamydia Bn9 DNA libraries were prepared using a TruSeq DNA Sample Kit (Illumina, San Diego, CA). Sequencing runs for paired-end sequences were performed using an Illumina Genome Analyzer (Illumina Hiseq, Illumina). The sequencing runs and read assembly of the libraries were carried out by Hokkaido System Science (Sapporo, Japan). Annotation of genes from the draft genome sequence was performed using Rapid Annotation using Subsystem Technology (RAST: http://rast.nmpdr.org/) [37 (link)] with a local manual BLASTp search.
Yamane C., Yamazaki T., Nakamura S., Matsuo J., Ishida K., Yamazaki S., Oguri S., Shouji N., Hayashi Y., Yoshida M., Y.i.m.i.n, & Yamaguchi H. (2015). Amoebal Endosymbiont Parachlamydia acanthamoebae Bn9 Can Grow in Immortal Human Epithelial HEp-2 Cells at Low Temperature; An In Vitro Model System to Study Chlamydial Evolution. PLoS ONE, 10(2), e0116486.
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6

RNA-Seq Analysis of TRAP Samples

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RNA purification from TRAP samples and from 5% of their corresponding unbound fractions was performed using RNeasy Mini Kit (Qiagen) following manufacturer’s instructions and used for subsequent sequencing. RNA integrity was assayed by using an RNA Pico chip on Bioanalyzer 2100 using 2100 Expert Software (Agilent, Santa Clara, CA) and only samples with RIN>9 were considered for subsequent analysis. Double-stranded cDNA was generated from 1-5 ng RNA using Nugen Ovation V2 kit (NuGEN, San Carlos, CA) following manufacturer’s instructions. 500 ng of cDNA per sample was sonicated to obtain fragments of 200 base pairs using Covaris-S2 system (Duty cycle: 10%, Intensity: 5.0, Bursts per second: 200, Duration: 120 seconds, Mode: Frequency sweeping, Power: 23W, Temperature: 5.5°C - 6°C, Covaris Inc., Woburn, MA). These fragments were used to produce libraries for sequencing by TruSeq DNA Sample kit (Ilumina, San Diego, CA, USA) following manufacturer’s instructions. The quality of the libraries was assessed by the 2200 TapeStation (Agilent). Multiplexed libraries were directly loaded on NextSeq 500 (Ilumina) with High Output single read sequencing using 75 cycles. Raw sequencing data was processed with Illumina bcl2fastq2 Conversion Software v2.17.
Badimon A., Strasburger H.J., Ayata P., Chen X., Nair A., Ikegami A., Hwang P., Chan A.T., Graves S.M., Uweru J.O., Ledderose C., Kutlu M.G., Wheeler M.A., Kahan A., Ishikawa M., Wang Y.C., Loh Y.H., Jiang J.X., Surmeier D.J., Robson S.C., Junger W.G., Sebra R., Calipari E.S., Kenny P.J., Eyo U.B., Colonna M., Quintana F.J., Wake H., Gradinaru V, & Schaefer A. (2020). Negative feedback control of neuronal activity by microglia. Nature, 586(7829), 417-423.
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7

RNA-seq Analysis of I-BET858 Treatment

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Double-stranded cDNA was generated from 1–5 ng purified RNA using Ovation V2 kit (NuGEN) following manufacturer’s instructions. 500 ng of cDNA per sample were sonicated to obtain fragments of 200 bp using Covaris-S2 system (duty cycle,10%; intensity, 5.0; Bursts per second, 200; duration, 120 s; mode, frequency sweeping; power, 23 W; temperature, 5.5–6°C; Covaris Inc.). These fragments were then used to produce sequencing libraries with the TruSeq DNA Sample kit (Illumina). The quality of the libraries was ensured using the 2200 TapeStation (Agilent). Duplexed libraries were sequenced on HiSeq 2000, typically yielding on average 60 million, 100-bp-long single-end reads per sample (Illumina). All samples were mapped at a rate of 79–80%. After filtering out adaptor and low-quality reads, reads were mapped using TopHat (version 2.0.8) to the mm9 mouse genome. The Cufflinks/Cuffdiff suite was used to estimate gene-level expression values as fragments per kilobase of exon model per million mapped fragments (FPKM). Differentially expressed autosomal genes between control and acute I-BET858 or chronic I-BET858 libraries were determined using a p-value <0.05 and fold change >2. Values with a FPKM <0.5 were excluded.
Sullivan J.M., Badimon A., Schaefer U., Ayata P., Gray J., Chung C.W., von Schimmelmann M., Zhang F., Garton N., Smithers N., Lewis H., Tarakhovsky A., Prinjha R.K, & Schaefer A. (2015). Autism-like syndrome is induced by pharmacological suppression of BET proteins in young mice. The Journal of Experimental Medicine, 212(11), 1771-1781.
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