0·5-2 × 106 cells from each sample were stored in 1 mL TRIzol reagent (Invitrogen cat. 15,596,026). RNA was isolated by the Washington University Tissue Procurement Center and sequenced by the Washington University Genome Technology Access Center on an Illumina Hi-Seq 3500 to generate 50 bp single-end reads. Reads were aligned to hg19 with UCSC annotations using STAR (v2·5·3a) [26 (link)]. RPKM normalized genome browser tracks were created with deepTools' (v3·1·0) bamCoverage utility and visualized on the UCSC genome browser. Read quantification was performed by Salmon (v0·11·0) using UCSC hg19 knownGene annotations [27 (link)], and differential gene expression analyses were done with the DESeq2 R package (v1·20·0) [28 (link)]. Genotify (v1·2·1) was used for manual gene curation [29 ]. All data analysis was done in SoS Notebook environments [30 ]. All gene ontology and pathway enrichments were performed on the Enrichr web server [31 (link)].
Hiseq 3500
Manufactured by Illumina
Sourced in China
The HiSeq 3500 is a high-throughput DNA sequencing system developed by Illumina. It is designed to generate large volumes of sequencing data with high accuracy. The HiSeq 3500 utilizes Illumina's proprietary sequencing-by-synthesis technology to perform DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Ab4729, by Abcam (1 mentions)
Rnaclean beads, by Beckman Coulter (1 mentions)
Nextera xt kit, by Illumina (1 mentions)
Ovation rna seq system v2 kit, by Tecan (1 mentions)
Dynabeads oligo dt magnetic beads, by Thermo Fisher Scientific (1 mentions)
Fragmentation buffer, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
10 protocols using hiseq 3500
1
RNA-seq profiling of cell samples
2
Chromatin immunoprecipitation and sequencing
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0.5–1.0 × 105 cells were snap-frozen for 15 min on dry ice and stored at −80 °C until use. Ultra-low-input chromatin immunoprecipitation for H3K9/K14 ac (EMD-Millipore 06–599) and H3K27ac (Abcam ab4729) was performed as described [16 (link)]. DNA was sequenced by the Washington University Genome Technology Access Center on an Illumina Hi-Seq 3500 to generate 50 bp single-end reads. Reads were aligned to hg19 with bowtie2 (v2·2·5) with default settings [17 (link)]. Reads in ENCODE blacklisted regions were removed with samtools (v1·3) [18 (link),19 (link)]. Peaks were called with MACS2 (v2·1·0·20150420) with the settings –q 0·01 –m 10 50 ‐‐nomodel ‐‐shiftsize = 150 and input controls [20 ]. RPKM normalized genome browser tracks were created with deepTools' (v3·1·0) bamCoverage utility with settings ‐‐binSize 10 ‐‐extendReads 150 ‐‐normalizeUsing RPKM and visualized on the UCSC genome browser [21 (link),22 (link)]. ChIPQC (v1·14·0) was used for quality control [23 ], and samples with fewer than 30% (H3ac) or 25% (H3K27ac) reads in peaks were removed from subsequent analyses. The DiffBind R package (v2·8·0) was used to derive consensus peak sets and determine differentially bound peaks between sample groups [24 (link)]. The chipSeeker R package (v1·16·1) was used to annotate peaks [25 (link)].
3
Plasmid Isolation and Sequencing of RBD Variants
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Plasmid samples were prepared from 30 OD units (1.6e8 cfu) of pre-selection yeast populations and approximately 5 OD units (∼3.2e7 cfu) of overnight cultures of plasma-escaped cells (Zymoprep Yeast Plasmid Miniprep II) as previously described (Greaney et al., 2021 (link)). The 16-nucleotide barcode sequences identifying each RBD variant were amplified by PCR and prepared for Illumina sequencing as described in (Starr et al., 2020 (link)). Barcodes were sequenced on an Illumina HiSeq 3500 with 50 bp single-end reads. To minimize noise from inadequate sequencing coverage, we ensured that each antibody-escape sample had at least 2.5x as many post-filtering sequencing counts as FACS-selected cells, and reference populations had at least 2.5e7 post-filtering sequencing counts.
4
Single-Cell RNA-seq Workflow
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Single cells were sorted directly into 96 well plates with lysis buffer. Single cell lysates were then converted to cDNA following capture with Agencourt RNA Clean beads using the SmartSeq2 protocol as previously described [79 (link)]. The cDNA was amplified using 20–24 PCR enrichment cycles prior to quantification and dual-index barcoding with the Illumina Nextera XT kit. The libraries were enriched with 12 cycles of PCR, then combined in equal volumes prior to final bead cleanup and sequencing. All libraries were sequenced on an Illumina HiSeq 3500 by either single-end 150 bp reads or short paired-end reads. Alignment was performed using STAR version 2.5.2b and transcripts were annotated using MacaM Rhesus genome assembly and annotation (v7.8.2: https://www.unmc.edu/rhesusgenechip/index.htm#NewRhesusGenome ) [80 (link)]. Transcript abundance estimates were calculated internal to the STAR aligner using the algorithm of htseq-count [81 (link)].
5
Tibetan Sheep Rumen Epithelial miRNA Libraries
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To construct cold-season (n = 3) and warm-season (n = 3) Tibetan sheep rumen epithelial miRNA libraries, 1 μg of total RNA from each sample with satisfactory quality testing (concentration ≥ 200 ng/μL, OD260/280 between 1.7–2.5, OD260/230 between 0.5–2.5, RIN value ≥ 8) was selected as the starting RNA amount for library construction. The library construction kit (VAHTSTM Small RNA Library Prep Kit for Illumina, NR801-02, Vazyme, Nanjing, China) was then used to attach a universal splice to the 3′ and 5′ ends of small RNA. The first strand cDNA was then synthesized by reverse transcription, and the product was purified by PCR amplification using VAHTSTM DNA Clean Beads (N411-03), followed by polyacrylamide gel electrophoresis (PAGE), gel cutting and gel recovery to finally obtain the cold and warm season Tibetan sheep rumen epithelial miRNAs. A sheep rumen epithelial miRNA library was obtained. The sequencing process was completed by Biomarker Technologies Corporation (Beijing, China) using Illumina HiSeq3500, and raw sequenced sequences (raw reads) were obtained.
6
SARS-CoV-2 RBD Variant Sequencing
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Plasmid samples were prepared from overnight cultures of antibody-escaped and 30 OD units (1.6e8 cfus) of pre-selection yeast populations (Zymoprep Yeast Plasmid Miniprep II). The 16-nucleotide barcode sequences identifying each RBD variant were amplified by PCR and prepared for Illumina sequencing exactly as described in Starr et al. (2020) (link). Barcodes were sequenced on an Illumina HiSeq 3500 with 50 bp single-end reads. To minimize noise from inadequate sequencing coverage, we ensured that each antibody-escape sample had at least 3x as many post-filtering sequencing counts as FACS-selected cells, and reference populations had at least 2.5e7 post-filtering sequencing counts.
7
RAD-seq Genotyping of Atlantic Salmon
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DNA samples were extracted from the 116 individuals using Macherey-Nagel NucleoSpin Tissue kit and quantified by fluorescence using Qubit 2.0. Sex of individuals was obtained through amplification and gel visualization of the sexually dimorphic sdY locus (Quéméré et al. 2014 (link)). Sbf1 restriction enzyme was used in the library preparation, and the single-end sequencing was performed on two lanes on an Illumina HiSeq 3500 platform. Final DNA concentration measures, library preparation and sequencing were obtained from a commercial provider, Plateforme MGX - Montpellier GenomiX (Montpellier, France) that delivered the sequences in FastQ format for bioinformatics analyses. Process_radtags function in Stacks v1.40 software (Catchen et al. 2013 (link)) was used to demultiplex, quality check and clean the data. Reads of 120 base pairs (average of 1643201 reads per individual, average 13.2X coverage Table S1) were aligned to the Atlantic salmon genome (GenBank: GCA_000233375.4) (Lien et al. 2016 (link)) using bowtie2 v2.3 (-p2, -sensitive, other parameters at default) (Langmead and Salzberg 2013 (link)).
8
Barcode Sequencing of Antibody-Escaped Variants
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Plasmid samples were prepared from overnight cultures of antibody-escaped and 30 OD units (1.6e8 cfus) of pre-selection yeast populations (Zymoprep Yeast Plasmid Miniprep II). The 16-nucleotide barcode sequences identifying each RBD variant were amplified by PCR and prepared for Illumina sequencing exactly as described in Starr et al. (2020) (link). Barcodes were sequenced on an Illumina HiSeq 3500 with 50 bp single-end reads. To minimize noise from inadequate sequencing coverage, we ensured that each antibody-escape sample had at least 3x as many post-filtering sequencing counts as FACS-selected cells, and reference populations had at least 2.5e7 post-filtering sequencing counts.
9
Transcriptomic Profiling of PDAC and PanIN
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Human primary PDAC and low-grade PanIN samples were collected from a total of 223 patients with PDAC who underwent surgery at the Columbia Pancreas Center. Frozen pancreas tissue banked at the Columbia University Medical Center were used. Only samples with a PDAC diagnosis for which intact RNA was available were selected. The diagnosis of all samples was confirmed by an independent gastro-intestinal pathologist prior to microdissection. For each tissue sample analyzed, epithelial and stromal cells were micro-dissected and isolated, yielding matched pairs for each patient. Whole transcriptome RNA amplification using the NuGEN Ovation RNA-Seq System V2 kit was performed on total RNA and yielded several µg of cDNA. The cDNAs were sequenced on an Illumina HiSeq 3500 to a depth of 30 million 100 bp single-end reads. In total, 197 epithelial with 100 matching stromal samples from primary PDACs and 26 epithelial with 23 matching stromal samples from low-grade PanIN were used in this study. Using GraphPad Prism, the data were analyzed using box and whisker plots to represent the medium, the interquartile, and the total range. Statistical significance between means was established via a two-way ANOVA with Tukey’s multiple comparison test (p < 0.05 [*], p < 0.01 [**], p < 0.001 [***]).
10
Transcriptome Profiling Using Illumina Sequencing
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The total RNA was extracted from cells using Trizol reagent following the manufacturer’s instructions (Invitrogen, USA). After the quality control of RNA, mRNA was enriched by Dynabeads® oligo dT magnetic beads (Thermo Fisher Scientific, USA), and then broke into shot fragments by fragmentation buffer (Agilent Technologies, California, USA). Afterward, the RNA fragments were reverse transcribed into the first strand cDNA with random hexamers. The second strand cDNA was compounded by adding into buffer, dNTPs, RNase H and DNA polymerase I. The final cDNA library was constructed after double strands cDNA were purified and repaired. The concentration of cDNAs in the library was attenuated into 1 ng/µL with a Qubit 2.0 fluorometer, and then cDNAs were detected using the Agilent Bioanalyzer 2100 (Agilent Technologies, California, USA). The libraries were pooled according to the data size and effective cDNA concentration. Finally, the cDNA libraries were sequenced on an Illumina HiSeq™ 3500.
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