Total RNA isolation and RNA sequencing were performed as previously described (113 (link)): RNA was extracted using the ARCTURUS PicoPure RNA Isolation Kit (Life Technologies) including a DNase treatment step (Qiagen) following manufacturer’s instructions. cDNA was generated with 1 ng of total RNA and thirteen cycles of amplification using the SMARTer Ultra Low RNA Kit for Illumina sequencing (Clontech) according to manufacturer’s instructions. The sequencing libraries were generated using the NEB Next ChIP-Seq kit (New England Biolabs) according the manufacture’s instructions. The quality of total RNA, cDNA and library were monitored throughout the process using a 2100 Bioanalyzer (Agilent). Sequencing was done in a HiSeq2000 sequencer (Illumina) with three samples per lane, paired-end 100bp.
Dnase treatment step
Manufactured by Qiagen
Sourced in Germany
The DNase treatment step is a laboratory technique used to remove DNA from RNA samples. It is a core function performed to ensure the purity and integrity of RNA preparations by eliminating any contaminating DNA.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000 sequencer, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Smarter ultra low rna kit for sequencing, by Illumina (1 mentions)
Nebnext chip seq kit, by New England Biolabs (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Amperase ung, by Thermo Fisher Scientific (1 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (1 mentions)
Quantstudio 6, by Thermo Fisher Scientific (1 mentions)
7500 real time system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini or micro kit, by Qiagen (1 mentions)
Biorobot ez1, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
Bioanalyzer 2100 rna 6000 nano kit, by Agilent Technologies (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer, by Cytek Biosciences (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
5 protocols using dnase treatment step
1
Total RNA Isolation and RNA Sequencing
2
Quantitative qPCR Analysis of Immune Genes
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Tissue samples from tumors and purified cells were kept frozen (−80°C) until mRNA extraction. As needed, samples were disrupted with a TissueLyser and homogenized in RLT buffer (Qiagen). RNA extraction was performed using the micro or mini RNeasy kit (Qiagen) using the DNAse treatment step (Qiagen), and cDNA preparation were conducted following standard procedures using the PrimeScript first-strand cDNA Synthesis Kit (Takara). Quantitative PCR was performed using TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG (Life Technologies) on the 7500 Real-Time System or QuantStudio 6 (Applied Biosystems) as indicated by the manufacturer. Primers and probes for the quantitative qPCR were analyzed with the following assay: GAPDH (Mm99999915_g1), IFNa4 (Mm00833969_s1_m1), IFNb (Mm00439552_s1), XCL1 (Mm00434772_m1), CXCL1 (Mm04207460_m1), CXCL9 (Mm00434946_m1), CCL5 (Mm01302427_m1), TNFα (Mm00443258_m1), IFNg (Mm01168134_m1), Nos2 (Mm01309897_m1), Perforin 1 (Mm00812512_m1), klrk1 (Mm01183328_m1), Rae (Mm00558293_g1), IL18 (Mm00434226_m1), H60a (Mm01311160_m1), IL12a (Mm00434169_m1), and H2K1 (Mm01612247_mH). All primers were obtained from Life Technologies.
3
Transcriptome Analysis of Head Kidney in Fish
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Each head kidney was carefully homogenized before RNA extraction using a Precellys 24 homogenizer and ceramic beads CK28 (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was extracted from the head kidney samples using the BioRobot EZ1 and QIAzol Lysis Reagent, with DNase treatment step (Qiagen, Germany). RNA yield was quantified with a NanoDrop® ND-1000 UV–Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA integrity assessed with a Bioanalyzer 2100 RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA). A 260/280 nm absorbance ratio of 1.8–2.0 indicates a pure RNA sample. All samples had an RNA integrity number (RIN) > 9.4. Library preparation and paired-end RNA-sequencing were carried out at the Norwegian High-Throughput Sequencing Centre (www.sequencing.uio.no ). Briefly, libraries were prepared with the TruSeq Stranded mRNA kit from Illumina (San Diego, CA, USA) which involves Poly-A purification to capture coding as well as several non-coding RNAs. The prepared samples were then sequenced on a NovaSeq S1 Flowcell sequencer (Illumina) at an average depth of 50 million reads per sample using a read length of 150 base pairs and an insert size of 420 base pairs.
4
Gene and microRNA Expression Analysis
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Cell pellets were lysed in Trizol® reagent and stored at −80°C. Total RNA was isolated by column extraction with a DNase treatment step (Qiagen, Valencia, CA). RNA was quantified using a Nanodrop 2000. For gene expression assays, RNA was reverse transcribed using MMLV reverse transcriptase (Invitrogen). Individual gene expression assays were done using specific TaqMan® probes and normalized to the endogenous control gene 18S. For microRNA expression assays, microRNA‐specific reverse transcription primers were multiplexed and RNA was reverse transcribed following the TaqMan® MicroRNA Reverse Transcription kit protocol. MicroRNA expression was normalized to RNU48, an abundant small‐nuclear RNA. Fold changes were calculated according to the ΔΔCt method.
5
Buffy Coat Isolation and RNA Extraction
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Whole blood was collected from 5 donors in Streck BCT®. Specimens were shipped at ambient temperature to our centralized clinical testing laboratory and processed within 4 days after blood was drawn. Whole blood was centrifuged at 800 ×g for 20 min, and buffy coat cells were recovered from the plasma/red blood cell (RBC) interface and purified using RBC Lysis Buffer (ammonium chloride-based lysing reagent), according to the manufacturer’s technical data sheet (Tonbo Biosciences, CA). RNA was extracted from purified buffy coat cells using the RNeasy® FFPE Kit, including a DNase treatment step (Qiagen, CA). The specimens used in our study were Institutional Review Board waived as they were remnant, de-identified specimens.
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