Total RNA was extracted from ~3 week cultured Neurons and DPSC using the miRNeasy Mini kit (Qiagen). Total RNA was checked for quantity and quality on an Agilent Bioanalyzer 6000 pico chip and determined to have an RNA Integrity Number (RIN) of ≥8.0. NuGen amplified material was sheared on a Covaris S2 with a duty cycle of 10%, intensity of 5100 cycles/burst, and 6 × 60 sec cycles (total processing time 6 min). 500ng of this sheared double strand DNA was then used to prepare libraries for sequencing using the Ion plus Core Library Module for AB Library Builder System. Libraries were used from this point without amplification. Before sequencing, small aliquots of this material were pooled and sequenced on an Ion Torrent PGM 314 chip. Barcode quantification data from the PGM were used to balance the samples for final pooling before sequencing. Following this final pooling the library pools were sized to a target size of 260 bp on a Pippin Prep instrument. The sized libraries were examined on an Agilent High Sensitivity DNA chip, quantified using real-time PCR, and used to prepare spheres using a One-Touch 2 device. These spheres were then sequenced on an Ion Torrent Proton sequencer with a P1 chip. On average, ~30 million reads were produced per sample for downstream analysis resulting in ≥7.6 million mapped reads per sample.
Bioanalyzer 6000 pico chip
Manufactured by Agilent Technologies
The Bioanalyzer 6000 pico chip is a lab equipment product by Agilent Technologies. It is designed to analyze and quantify nucleic acid samples, such as DNA and RNA, in a automated and high-throughput manner.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext ultra or ultra 2 directional rna kit, by New England Biolabs (2 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Pgm 314 chip, by Thermo Fisher Scientific (1 mentions)
Proton sequencer, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Hiseq instrument, by Illumina (1 mentions)
Phosflow protocol, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (1 mentions)
5 protocols using bioanalyzer 6000 pico chip
1
RNA-Seq Library Preparation for Neurons and DPSC
2
Transcriptome Analysis via mRNA-seq
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RNA was extracted from cells using TRIreagent (Sigma) following manufacturer’s instructions and RNA integrity assessed using a Bioanalyzer 6000 pico chip (Agilent). mRNA-seq libraries were prepared using the NEBNext Ultra (or Ultra II) Directional RNA Kit (NEB) with the NEBNext Poly(A) mRNA Magnetic Isolation Module and processed for sequencing as previously described in (39 (link)).
3
RNA-seq Analysis of Leishmania tarentolae
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RNA was isolated using TRIzol (Thermo Fisher Scientific) and resuspended in 10 mM Tris (pH 7), and RNA quality was assessed using the Bioanalyzer 6000 Pico chip (Agilent). mRNA was isolated from 1 μg total RNA using the New England BioLabs (NEB) poly(A) mRNA magnetic isolation module and prepared using the stranded RNA-seq protocol (67 (link)), modified for Leishmania as described previously (68 (link)). Libraries were sequenced on an Illumina HiSeq instrument, obtaining paired-end 150-bp reads. Reads were aligned against our in-house L. tarentolae genome with Bowtie2 (69 (link)) using the “very high sensitivity” parameter or the Geneious assembler (Geneious Prime 11.05) using the “low sensitivity/fastest” option. Differential expression analysis was performed on the Geneious assemblies using the DESeq2 module to compare Tet+ and Tet− samples from days 2, 3, 4, 6, and 8 for replicate 1 and from days 2, 3, 4, 5, 7, and 9 for replicate 2. Strand-specific read coverage was calculated directly from BAM files of the Bowtie2 alignments using customized pysam scripts (https://github.com/pysam-developers/pysam ).
4
RNA Extraction and mRNA-Seq Library Prep
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RNA was extracted from cells using TRIreagent (Sigma) following manufacturer's instructions and RNA integrity assessed using a Bioanalyzer 6000 pico chip (Agilent). mRNA-seq libraries were prepared using the NEBNext ultra (or ultra II) Directional RNA kit (NEB) with the NEBNext Poly(A) mRNA Magnetic Isolation Module.
5
Comprehensive Immune Cell Profiling Protocol
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PBMCs (0.3x10 6 ) were incubated with Fc-receptor blocking reagent (Miltenyi Biotec) followed by staining for surface markers (Supplementary Table S2). Intracellular staining was performed using the Cytofix/Cytoperm kit (BD Biosciences). Phosphorylation staining (2x10 6 PBMCs) was performed using the Phosflow protocol (BD Biosciences). Cell sorting was performed on a FACSAria III instrument. Representative gating strategy is depicted in Supplementary Figure S3.
The number and purity of NK cells (median purity 99%) used in the RNA-seq is enlisted in Supplementary Table S4. RNA was extracted from purified NK cells (Qiagen RNeasy Micro Kit) and RNA integrity and quantity were checked with the Bioanalyzer 6000 pico chip (Agilent). Whole-transcriptome analysis with next-generation sequencing (RNA-seq), preparative techniques and statistical comparison (21) were performed by the VIB Nucleomics Core (KU Leuven, www.nucleomics.be). Detailed methods can be found in Supplementary File S5.
The number and purity of NK cells (median purity 99%) used in the RNA-seq is enlisted in Supplementary Table S4. RNA was extracted from purified NK cells (Qiagen RNeasy Micro Kit) and RNA integrity and quantity were checked with the Bioanalyzer 6000 pico chip (Agilent). Whole-transcriptome analysis with next-generation sequencing (RNA-seq), preparative techniques and statistical comparison (21) were performed by the VIB Nucleomics Core (KU Leuven, www.nucleomics.be). Detailed methods can be found in Supplementary File S5.
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