The cDNA for paired-end RNA-seq analysis using a Genome Analyzer II (Illumina) was performed basically as described in the manufacturer's protocol (Illumina RNA-seq PE Sample Prep protocol, v3.6). First, 100 ng of cyanoethylated poly(A)+ RNA was fragmented in 20 μL of 1× fragmentation buffer at 95°C for 5 min, and RNA fragments were recovered by ethanol precipitation. First strand cDNA synthesis was performed with random primer at 25°C for 10 min then 50°C for 50 min. Subsequently, second strand cDNA synthesis was performed with RNase H and DNA polymerase I. Purified cDNA was end-repaired and ligated with adaptors. After that, the 300-bp cDNA was purified by 2% agarose gel electrophoresis and then amplified by PCR with 20 cycles. The amplified cDNA was subjected to 2% agarose gel electrophoresis, and the 300-bp cDNA band was cut and extracted from the gel. The size and quality of the cDNA was confirmed using a DNA 1000 kit and Bioanalyzer (Agilent). The cDNAs for CE−, CE+, and CE++ conditions were then subjected to a Genome Analyzer II (Illumina) according to the manufacturer's protocol.
Genome analyzer 2
Manufactured by Illumina
Sourced in United States, China, France, United Kingdom
The Genome Analyzer II is a high-throughput DNA sequencing system developed by Illumina. It utilizes sequencing-by-synthesis technology to determine the nucleotide sequence of DNA samples. The core function of the Genome Analyzer II is to perform massively parallel DNA sequencing, generating large volumes of genomic data for various applications, such as genetic research, diagnostics, and clinical studies.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (39 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions)
Trizol, by Thermo Fisher Scientific (8 mentions)
Bioanalyzer, by Agilent Technologies (7 mentions)
2100 bioanalyzer, by Agilent Technologies (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Rneasy plant mini kit, by Qiagen (5 mentions)
Klenow and dna prime labeling system, by Thermo Fisher Scientific (5 mentions)
Truseq rna sample preparation kit, by Illumina (5 mentions)
Genome analyzer, by Illumina (4 mentions)
Casava 1, by Illumina (4 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Gel extraction kit, by Qiagen (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (3 mentions)
Minelute pcr purification kit, by Qiagen (3 mentions)
Bioruptor, by Diagenode (3 mentions)
Pcr purification kit, by Qiagen (3 mentions)
Sonifier 250, by Emerson (3 mentions)
Protein a g agarose beads, by Merck Group (3 mentions)
Methylminer methylated dna enrichment kit, by Thermo Fisher Scientific (3 mentions)
303 protocols using genome analyzer 2
1
Paired-end RNA-seq Analysis using Illumina
2
Comparative RNA-seq Analysis of Lymphoblastoid and Burkitt's Lymphoma Cell Lines
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RNA-seq data sets from 22 Human B-Cell samples (lymphoblastoid cell lines [LCLs]) immortalized with Epstein-Barr Virus (EBV) were downloaded from the NCBI Sequence Read Archive (SRA010302). Samples were sequenced using an Illumina Genome Analyzer II machine running single end 50 base sequencing reactions. Similarly, 22 Human Burkitt's Lymphoma (BL) samples were obtained from the NCBI Sequence Read Archive (SRA048058). Samples were sequenced using an Illumina Genome Analyzer II machine running paired end 107 and 102 base sequencing reactions. The Akata RNA-seq data set was generated previously in our lab (SRA047981) [10] (link). The Akata sample was sequenced using an Illumina HiSeq instrument running paired end 100 base sequencing reactions. A list of all the samples used is provided in Table S1 .
3
Small RNA Sequencing and Annotation
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Small RNA libraries were run on an Illumina Genome Analyzer II (single end 36). After removing adapters, small RNA reads were first mapped to the mm9 mouse genome with Bowtie (Langmead et al. 2009 (link)), with up to two mismatches and a maximum of 100 multiple alignments (otherwise suppressed). Unmapped reads were remapped using STAR (Dobin et al. 2013 (link)) with the same criteria in order to extract spliced reads. Transcriptome and 5′RACE libraries were run on an Illumina Genome Analyzer II or MiSeq (single end 76 or single end 101). NSR transcriptome libraries were reverse-complemented and trimmed (removing the first 8 nt). They were then mapped to the mm9 mouse genome with STAR (allowing two mismatches and a maximum of 100 multiple alignments). 5′RACE libraries were mapped to the mm9 mouse genome with STAR (allowing two mismatches and a maximum of 100 multiple alignments).
All reads were annotated based on genomic locations against structural RNAs (University of California at Santa Cruz [UCSC] RepeatMasker track), microRNA (miRNA) (miRBase 18), TEs (UCSC RepeatMasker track), 214 pachytene piRNA cluster coordinates (Li et al. 2013 (link)), RefSeq genes (UCSC RefSeq track), and testis-specific lncRNAs (Liu et al. 2011 (link); Sun et al. 2013 (link)). At least 50% of the read was required to overlap with the feature before being assigned an annotation.
All reads were annotated based on genomic locations against structural RNAs (University of California at Santa Cruz [UCSC] RepeatMasker track), microRNA (miRNA) (miRBase 18), TEs (UCSC RepeatMasker track), 214 pachytene piRNA cluster coordinates (Li et al. 2013 (link)), RefSeq genes (UCSC RefSeq track), and testis-specific lncRNAs (Liu et al. 2011 (link); Sun et al. 2013 (link)). At least 50% of the read was required to overlap with the feature before being assigned an annotation.
4
sRNA Library Preparation and Sequencing
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The first set of sRNA libraries was done as described before [30 (link)] using total RNA from T. pseudonana grown in normal conditions, iron- and silicon- limitation as well as alkaline pH [11 (link)]. Briefly, the RNA was run in a 15% polyacrylamide gel, and the area corresponding to 19–24 nt length was excised and eluted. The RNA was ligated to 5’- and 3’-RNA adaptors, and after each ligation the products were gel-purified to eliminate non-ligated adaptors. RNA was converted to DNA by RT-PCR, and sequenced (36 cycles) using an Illumina Genome Analyzer II at BaseClear (Leiden, The Netherlands).
Total RNA from T. pseudonana and F. cylindrus was used for the second set of libraries, using the small RNA v1.5 kit from Illumina according to manufacturer’s recommendations. With this protocol it is not necessary to gel-purify the ligation products after each ligation. RNA was ligated to the adaptors, reverse transcribed, PCR amplified and purified from a polyacrylamide gel. cDNA libraries were sequenced (42 cycles) using an Illumina Genome Analyzer II at TGAC (Norwich, UK).
Total RNA from T. pseudonana and F. cylindrus was used for the second set of libraries, using the small RNA v1.5 kit from Illumina according to manufacturer’s recommendations. With this protocol it is not necessary to gel-purify the ligation products after each ligation. RNA was ligated to the adaptors, reverse transcribed, PCR amplified and purified from a polyacrylamide gel. cDNA libraries were sequenced (42 cycles) using an Illumina Genome Analyzer II at TGAC (Norwich, UK).
5
Comparative Analysis of Human and Mouse DNA Methylomes
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The BS-seq data of IMR90 and H1 were obtained from the human DNA methylome database of Salk Institute [57 ]. Four samples (two replicates each) were sequenced on an Illumina Genome Analyzer II with an average depth of 14.5× per strand, yielding 1.16 and 1.18 billion reads for IMR90 and H1, respectively. The reads were originally mapped to the hg18 reference genome, covering 2464,851 CpGs in chromosome 1. The genome coordinates were then converted from hg18 to hg38 using the UCSC liftOver command line version. The mouse BS-seq data [43 (link)] were downloaded from the Gene Expression Omnibus (accession: GSE33722). Two reciprocal crosses were sequenced on an Illumina Genome Analyzer II with an average depth of 23.75×, generating 1.54 billion and 1.33 billion reads for F1i and F1r, respectively. The reads were mapped to the mm9 reference genome, covering 11,345,372 CpG dinucleotides.
6
Evaluating Squeegee's Contamination Detection Capabilities
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To further demonstrate the contamination detection capabilities of Squeegee, we leveraged a human-derived RNA-seq dataset from a study performed by the Genetic European Variation in Health and Disease (GEUVADIS) consortium. The dataset contains parallel RNA-Seq samples from Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines that are sequenced across seven different sequencing centers with identical library preparation kits. We used samples from six out of seven sequencing centers that used the Illumina sequencing platform (Illumina Genome Analyzer II) and excluded the ones that used AB SOLiD System 3.0, leaving us with a total number of 40 paired-end sequencing runs.
We then mapped each of the sequencing runs with bowtie2 against the human reference genome (Homo sapiens GRCh38.p13) with the parameter –maxins 600 to remove human reads. We gathered the unmapped reads for each of the samples and used them as input for Squeegee. The sample type for each of the samples is labeled by the sequencing center where it was run. The parameter settings for Squeegee and data characteristics are shown in Supplementary Table1 .
We then mapped each of the sequencing runs with bowtie2 against the human reference genome (Homo sapiens GRCh38.p13) with the parameter –maxins 600 to remove human reads. We gathered the unmapped reads for each of the samples and used them as input for Squeegee. The sample type for each of the samples is labeled by the sequencing center where it was run. The parameter settings for Squeegee and data characteristics are shown in Supplementary Table
7
ChIP-seq analysis of GATA6-induced mES cells
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Gata6-inducible mES cells were seeded at 1 × 104 cells per square centimeter and treated with 1 µg/mL doxycycline for 36 h prior to harvesting. Immunoprecipitation was performed on 1 × 107 to 2 × 107 cells as described (Vokes et al. 2007 (link)) for three biological replicates versus input samples. Sonication was performed using a Misonix 4000 (28 cycles of 15 sec on and 45 sec off at an intensity of 70%) with a microtip probe (Misonix). The antibodies used are listed in Supplemental Table S7. Libraries were prepared using the TruSeq ChIP sample preparation kit, and the resulting samples were sequenced using the Illumina Genome Analyzer II (Illumina). Data will be deposited into Gene Expression Omnibus and released immediately after publication (GSE69323). Computational analysis details are included in the Supplemental Material.
8
Small RNA Sequencing of Grapevine Leaves
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The mixed leaf samples from ‘Summer Black’ grapevine at three different growth stages, young leaves (two weeks old), large leaves (five weeks old), and old leaves (nine weeks old) treated with CuSO4, were used for RNA extraction. The total RNA samples were first extracted using our modified CTAB method [21 (link)]. Isolation of small RNAs and preparation of small RNA libraries were performed based on the procedure of Wang et al. [21 (link)]. sRNAs were first separated from the total RNA by size fractionation with 15% TBE urea polyacrylamide gel (TBU) and small RNA regions corresponding to the 18–30 nucleotide bands in the marker lane were excised and recovered. The 18–30 nt small RNAs were 5′ and 3′ RNA adapter-ligated by T4 RNA ligase and at each step, length validated and purified by TBU electrophoretic separation. The adapter-ligated small RNA was subsequently transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen) and PCR amplified using primers that annealed to the ends of the adapters. The amplified cDNA constructs were purified and recovered. The purified cDNA from the two sRNA libraries were sequenced with an Illumina Genome Analyzer II (LC Sciences, Hangzhou, China) according to the manufacturer's protocols.
9
RNA Extraction and Sequencing Protocol
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Total RNA were extracted from pooled lateral bud samples using the EASYSPIN RNA extraction kit (Yuanpinghao Biotech Co.,Ltd) and treated with RNase-free DNase I for 30 min at 37 °C to remove residual DNA. RNA electrophoresis was run for an initial assessment of sample quality and quantity.
The mRNA was then purified from the total RNA samples and was used to synthesize cDNA. After end repair, adding dA and adapters, the cDNA products were further amplified by PCR using adapter primer sequences. The cDNA libraries were then quantified and sent to sequencing using Illumina Genome Analyzer II.
The mRNA was then purified from the total RNA samples and was used to synthesize cDNA. After end repair, adding dA and adapters, the cDNA products were further amplified by PCR using adapter primer sequences. The cDNA libraries were then quantified and sent to sequencing using Illumina Genome Analyzer II.
10
ChIP-seq Analysis of p300 Binding
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For p300, we chemically crosslinked and sonicated cells to generate fractionated genomic DNA. Chromatin immunoprecipitation was performed by using anti-p300 (sc-585, Santa Cruz Biotechnology). The DNA fragments were blunt-end ligated to the Illumina adaptors, amplified, and sequenced by using the Illumina Genome Analyzer II (Illumina, San Diego, CA). Sequence reads of 25 or 36 bps were obtained by using the Illumina Analysis Pipeline. Publically available ChIP-seq datasets are listed in Table S1 and were obtained from several published studies 12 (link)-15 (link),26 (link)-29 (link).
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