Dissociated iNIL‐MNs were plated on PLO‐coated 10‐cm culture dishes. Upon indicated times, cells were harvested using a cell scraper, and RNA was purified using the QIAshredder and RNAeasy extraction kits (Qiagen) according to the manufacturer's instructions. The quality of the RNA sample was monitored by Bioanalyzer (Agilent, Santa Clara, CA, USA), and the RNA samples were submitted to Columbia University Genome Center for RNA‐seq analysis. The list of differentially expressed genes was submitted to Qiagen Ingenuity Pathway Analysis to examine biological pathways altered by drug treatment.
Rnaeasy extraction kit
Manufactured by Qiagen
Sourced in Germany, United States
The RNAeasy extraction kit is a laboratory tool designed for the purification of RNA from a variety of biological samples. It utilizes a silica-membrane-based technology to efficiently capture and isolate RNA molecules.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Lightcycler 480, by Roche (5 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Qiashredder, by Qiagen (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Isol rna lysis reagent, by Thermo Fisher Scientific (2 mentions)
Isol rna lysis reagent, by Qiagen (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Tapesstation, by Agilent Technologies (2 mentions)
Novaseq 6000 sp 50 bp, by Illumina (2 mentions)
Ingenuity pathway analysis (ipa), by Qiagen (1 mentions)
Taqman rt kit, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sensifast sybr no rox kit, by Meridian Bioscience (1 mentions)
Iscript reverse transcription supermix, by Bio-Rad (1 mentions)
Taqman gene expression assays protocol, by Thermo Fisher Scientific (1 mentions)
54 protocols using rnaeasy extraction kit
1
Transcriptome Analysis of Dissociated iNIL-MNs
2
RNA Isolation and qPCR Analysis
3
RAS Isoform Gene Expression Analysis
4
Transcriptional Regulation of Carbon Concentrating Mechanisms
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HC, LC and VLC grown cells were pelleted for 2 min at 800 x g, the supernatant discarded, and the pellet flash frozen in liquid nitrogen. Total RNA was extracted using the Qiagen RNA easy extraction kit following the manufacturer’s instructions. cDNA was synthesized from total RNA using the iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA) with 1 µg of RNA in a 20 µL reaction volume. cDNAs were diluted to 50 µL and used as template for Real-Time PCR monitored by SensiFast SYBR No-Rox Kit (Bioline, Cincinatti, OH, USA). RT-PCR was performed in the Roche Light Cycler 480 using primers for CAH4, CCP1, LCIA, HLA3 and CBLP (Table 2 ).
5
Microarray Analysis of Tumor Transcriptomes
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Total RNA was isolated and purified from cells using Isol-RNA Lysis Reagent (Fisher.) Total RNA from tumors was isolated and purified from frozen specimens using Isol-RNA Lysis Reagent and Qiagen RNAeasy extraction kit with DNAase I on column treatment. Labeled RNA was hybridized to the Affymetrix GeneChip Mouse Gene 1.0 and 2.0 ST Array. Affymetrix CEL files for all samples were processed using the RMA method as implemented in the “oligo” R package version 1.26.6. Probe annotations were provided by the “mogene10sttranscriptcluster.db” and “mogene20sttranscriptcluster.db” R package version 8.0.1 and 2.13.0, respectively. Since different array types and different batches were used, each expression set was z-score transformed27 (link) and median centered. Multiple probes for the same gene were averaged and only genes common to the 1.0 and 2.0 ST arrays were kept. Batch effects were adjusted using the ComBat method as implemented in the “sva” R package version 3.8.0. The microarray data has been deposited at the GEO (GSE65503) and processed data provided as SI Table 2 . Gene expression data for primary melanoma samples were downloaded from the GEO (GSE22155). For this data set, the post-processed data and provided annotations were used.
6
Measuring PGC-1α and PPARα Gene Expression
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The expression of PGC-1α and PPARα genes was measured using RNA obtained from cell extracts (n = 7) with an RNAeasy extraction kit (Qiagen). An amount of 500 ng of RNA was reverse-transcribed to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems) in a thermal cycler at: 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. The cDNA was then transcribed to mRNA in a real-time PCR reaction according to the TaqMan gene expression assays protocol (Applied Biosystems) and was assayed with Taqman probes Mm00440939_m1, Mm00482488_m1, and Mm00607939_s1, which were used to detect PPARα, PGC-1α, and β-actin, respectively. Results are presented in relative mRNA units standardised to β-actin.
Phospho-p38 MAPK (Thr180/Tyr182) was assayed by a PathScan® Sandwich ELISA Kit #7946 according to the manufacturer’s instructions.
Phospho-p38 MAPK (Thr180/Tyr182) was assayed by a PathScan® Sandwich ELISA Kit #7946 according to the manufacturer’s instructions.
7
RNA Extraction and Library Preparation for Hepatic Transcriptome Sequencing
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Total RNA was extracted from hepatic tissue using an RNAeasy extraction kit from QIAGEN®30 with a DNAse cleaning step according to the manufacturer’s instructions. RNA quality was measured using Nanodrop, Qubit 2.0 fluorometer and Bioanalyzer 2100 instruments.
Fifteen libraries (one per individual) were constructed from 1.0 µl of total RNA using the KAPA Stranded RNA-Seq kit with RiboErase from KapaBiosystems®. Ribosomal RNA was removed by depletion by DNA primer hybridization followed by treatment with RNAse and DNAse according to the manufacturer’s instructions. The fragmentation cycle was adjusted to 10 cycles of amplification at 94 °C for 5 minutes to obtain fragments between 100 and 2100 bp in length. Each library was enriched with Illumina® TruSeq Index Adapters (250 nM) for multiplex sequencing.
Library quality was evaluated using a Qubit 2.0 fluorometer and an Agilent Bioanalyzer 2100; good quality libraries were paired-end (PE) sequenced on an Illumina HiSeq. 4000 at the Center for Genomics Services of the University of California, Berkeley.
Fifteen libraries (one per individual) were constructed from 1.0 µl of total RNA using the KAPA Stranded RNA-Seq kit with RiboErase from KapaBiosystems®. Ribosomal RNA was removed by depletion by DNA primer hybridization followed by treatment with RNAse and DNAse according to the manufacturer’s instructions. The fragmentation cycle was adjusted to 10 cycles of amplification at 94 °C for 5 minutes to obtain fragments between 100 and 2100 bp in length. Each library was enriched with Illumina® TruSeq Index Adapters (250 nM) for multiplex sequencing.
Library quality was evaluated using a Qubit 2.0 fluorometer and an Agilent Bioanalyzer 2100; good quality libraries were paired-end (PE) sequenced on an Illumina HiSeq. 4000 at the Center for Genomics Services of the University of California, Berkeley.
8
Quantitative RNA Editing Assessment
9
Transcriptomic Response of K. pneumoniae to L-Ag NPs
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A freshly grown culture of K. pneumoniae MGH78578 from mid-log10 phase (approximately 107 CFU mL–1) was exposed to MIC75 L-Ag NPs for 5 and 30 min at 37°C in mLB broth. Bacterial cells not exposed to L-Ag NPs were selected as a control for each time point. RNA was isolated using RNAeasy extraction kit (Qiagen) and treated with Turbo DNase kit (Ambion’s). RNA integrity was assessed using a Bioanalyzer 2100 RNA 6000 nanochip (Agilent Technologies). RNA library sequencing was performed at the Centre for Genomic Research, University of Liverpool, United Kingdom. Ribosomal RNA was removed with a Ribo-Zero rRNA removal kit (Illumina). Libraries were prepared with NEBNext Directional RNA Library Prep Kit (BioLabs). Pooled libraries (Supplementary Table 1 ) were loaded on cBot (Illumina) and cluster generations was performed. Single-sequencing using 150 bp read length was performed on lane of the HiSeq 4000 sequencer (Illumina). Raw sequencing data was processed as described in Supplementary Material . The RNAseq data produced from the present work were deposited to the NCBI-GEO database and are available under the accession number GSE151953 .
10
Profiling Gene Expression from Blood Samples
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Blood samples were stored at 4 °C on the day of collection until they were shipped overnight each day to our blood processing laboratory in Boston, MA in an insulated container with a cooler pack to keep samples chilled. Upon arrival, RNA was extracted using the Qiagen RNAEasy extraction kit, according to protocol and then stored at −80 °C until analysis. Gene expression profiling was conducted using the Illumina HumanHT-12 v4 Expression BeadChip, with RNA labeling and array hybridization performed according to protocol. Image capture was performed using the Illumina BeadArray Reader. Standard QC and preprocessing procedures were applied to remove failed samples (n = 2). Standard background correction and normalization procedures (Variance-Stabilizing Transform, [16 (link)]) were applied using the R package lumi. The final data set included information from 47,295 probes on 165 samples from 63 subjects.
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