We used the ARTseqTM kit and followed the kit protocol with minor modifications. Briefly, animal samples were thawed in lysis buffer, omitting cycloheximide to avoid a 5′ end bias. Lysates were clarified by centrifugation for 10 min at 20,000 x g at 4 °C. Amount of ARTseq nuclease was optimized, following steps in the ARTseq kit protocol, resulting in 2 units used to digest RNA. Monosomes were purified using MicroSpin S-400 columns and centrifugation for 2 min at 600 x g. Ribosome protected RNA fragments (RPFs) were purified using Zymo RNA Clean & Concentrator-25 kit. rRNA was removed using Ribo-Zero and RPFs PAGE purified, eluted and precipitated and resuspended in nuclease-free water and libraries prepared for sequencing following the kit protocol. Libraries were prepared in three biological replicates.
Rna clean concentrator 25 kit
Manufactured by Zymo Research
Sourced in United States, Germany
The RNA Clean & Concentrator-25 kit is a product offered by Zymo Research. It is designed to purify and concentrate RNA samples. The kit utilizes a selective binding to a silica-based column to capture RNA, allowing for efficient removal of contaminants and concentration of the RNA sample.
Automatically generated - may contain errors
Lab products found in correlation
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (7 mentions)
Turbo dnase, by Thermo Fisher Scientific (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by New England Biolabs (4 mentions)
Engen sgrna synthesis kit, by New England Biolabs (3 mentions)
Tri reagent, by Merck Group (3 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (3 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Dnase 1 amplification grade, by Thermo Fisher Scientific (2 mentions)
Rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Poly a polymerase tailing kit, by Illumina (2 mentions)
M7g 5 ppp 5 g, by New England Biolabs (2 mentions)
Hiscribe t7 arca mrna kit, by New England Biolabs (2 mentions)
2200 tapestation system, by Agilent Technologies (2 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
49 protocols using rna clean concentrator 25 kit
1
ARTseq-based Ribosome Profiling
2
Anaerobic Bacterial Culture and RNA Extraction
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Starting from fresh precultures, 20 mL bacterial cultures were carried out anaerobically in duplicates for 16 hours at 34 °C and 120 rpm, starting with an initial OD600 value of ≈0.1. Ten mL samples were stabilized on ice by addition of a cold stop solution (ethanol:acid phenol at 1:18 ratio), centrifuged, and the cell pellets were frozen at −80 °C.
TRI reagent (Ambion) was further used to lyse the cells. RNAs were then extracted with the Direct-zol RNA purification kit (Zymo Research). After DNase treatment (Turbo DNase, Invitrogen), RNAs were column-purified with the RNA Clean & Concentrator 25 kit (Zymo Research).
For reverse transcription, 100 ng or RNAs were used in 20 µL reactions (iScript cDNA Synthesis Kit, Bio-Rad). Of the resulting cDNAs, 0.5 µL were then used for qPCR (SsoAdvanced Universal SYBR Green Supermix, Bio-Rad) along with adequate controls (no RT controls, positive controls on gDNA, no template controls). Primers are given with their corresponding qPCR efficiency in Supplementary FileS6 .
qPCR data were analyzed with the ΔΔCq method, using gapC (CIBE_0769) as a housekeeping gene control and taking into account reaction efficiencies.
TRI reagent (Ambion) was further used to lyse the cells. RNAs were then extracted with the Direct-zol RNA purification kit (Zymo Research). After DNase treatment (Turbo DNase, Invitrogen), RNAs were column-purified with the RNA Clean & Concentrator 25 kit (Zymo Research).
For reverse transcription, 100 ng or RNAs were used in 20 µL reactions (iScript cDNA Synthesis Kit, Bio-Rad). Of the resulting cDNAs, 0.5 µL were then used for qPCR (SsoAdvanced Universal SYBR Green Supermix, Bio-Rad) along with adequate controls (no RT controls, positive controls on gDNA, no template controls). Primers are given with their corresponding qPCR efficiency in Supplementary File
qPCR data were analyzed with the ΔΔCq method, using gapC (CIBE_0769) as a housekeeping gene control and taking into account reaction efficiencies.
3
RNA Extraction and cDNA Synthesis
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Ten milliliters of cell cultures taken from exponentially growing strains was harvested and lysed with 1 ml of TRIzol reagent (Invitrogen, USA). Total RNA was extracted and isolated with a PureLink RNA Minikit (Thermo Fisher Scientific, USA) and then cleaned and concentrated with an RNA Clean & Concentrator-25 kit (Zymo Research, USA). To exclude potential DNA contamination, the total RNA (∼2 μg) was treated with 2 μl of amplification-grade DNase I (1 U/μl; Invitrogen, USA). Approximately 500 ng of DNase I-treated total RNA was reverse transcribed using 2 μM gene-specific reverse primers and 1 U of SuperScript IV reverse transcriptase (Thermo Fisher Scientific, USA) according to the manufacturer’s guidelines. Two microliters of synthesized cDNA was used as a template in a standard PCR amplification reaction performed in a thermocycler (Bio-Rad, USA), using Phusion High-Fidelity DNA polymerase (NEB, USA) and the gene-specific primers binding the regions inside the slaA and slaB genes (Table S3). Genomic DNA and total RNA were used as templates for positive and negative controls in PCR amplification, respectively. The resulting PCR products were separated by electrophoresis with a 1.2% (wt/vol) Tris-acetate EDTA (TAE) agarose gel.
4
RNA Extraction and Depletion for RNA-seq
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For samples GS271-GS276, total RNA was extracted using RNeasy Mini Kit (Qiagen, #74106) including on-column DNAse digestion (Qiagen, #79254). Ribosomal RNA was depleted using RNA Ribo-Zero rRNA Removal Kit (Illumina, #MRZH11124). For samples GS947-GS950, total RNA was extracted using Trizol and the RNA Clean & Concentrator -25 Kit (Zymo Research, #R1017) including on-column DNAse digestion (Qiagen, #79254). Due to the discontinued Ribo-Zero kit, ribosomal RNA was depleted with the NEBNext rRNA Depletion Kit (NEB, #E6350). Libraries were prepared with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, #E7420S). Sequencing was performed by LAFUGA on an Illumina Hiseq 1500 using 50 bp paired-end runs.
5
Generation of FMNL2-EGFP Fusion Construct
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Template RNA was generated from mouse ovaries with RNA Isolation Kit (Thermo Fisher), then we reversed transcription of these RNA to create cDNA by a PrimeScript 1st strand cDNA synthesis kit (Takara, Japan). Fmnl2-EGFP vector was generated by Wuhan GeneCreate Biological Engineering Co, Ltd. mRNA was synthesized from linearized plasmid using HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G (5′) ppp (5′) G (NEB) and tailed with a poly(A) polymerase tailing kit (Epicentre) and purified with RNA clean & concentrator-25 kit (Zymo Research).
6
In Vitro Transcription of RNA Aptamers
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Partially double-stranded DNAs or PCR products (46 (link)) were used as a template for in vitro transcription. RNA aptamers were synthesized by in vitro transcription at 42°C for 4 h using HiScribe T7 High Yield RNA Synthesis Kit (NEB) according to the manufacturer's manual for in vitro transcription of short templates (Supplementary Table S1 ). Transcription products were treated with 2 U TURBO DNase (Thermo Fisher Scientific) for 60 min at 37°C and purified with RNA Clean & Concentrator-25 Kit (Zymo Research). RNA concentrations were determined by absorbance at 260 nm according to OligoCalc (47 (link)).
7
Transcriptome Profiling of Rat and Mouse Tissues
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For mRNA-seq and totRNA-seq, total RNA was isolated using TRIzol Reagent (Invitrogen; 15596018) using 5–10 mg rat and mouse tissue of the exact same powdered tissue samples (from the exact same animals) used for Ribo-seq. RNA was DNase treated and purified using the RNA Clean & Concentrator™-25 kit (Zymo Research; R1018). RIN scores were measured on a BioAnalyzer 2100 using the RNA 6000 Nano assay (Agilent; 5067-1511). Poly(A)-purified mRNA-seq libraries or ribosomal RNA-depleted totRNA-seq libraries were generated from the same sample of high-quality RNA (average RNA integrity number (RIN) for HXB/BXH rats of 9.1 (Additional file 1 : Figure S1A). RNA-seq library preparation was performed according to the TruSeq Stranded mRNA or total RNA Reference Guide, using 500 ng of total RNA as input. Libraries were multiplexed and sequenced on an Illumina HiSeq 2500 or 4000 producing paired-end 2 × 101 nt reads.
8
RNA Extraction and Sequencing from Brain Tissue
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The brain tissues were dissected rapidly, flash-frozen in liquid nitrogen, and maintained on dry ice until storage at –80°. Frozen brain tissue was homogenized in 1 ml TRIzol solution (Life Technologies 15596-018), using a motorized homogenizer. Samples were then centrifuged at 4° for 10 min at 4000 rpm to separate out debris, and the resulting supernatant was processed for total RNA extraction. Total RNA was treated with TURBO DNase (Ambion AM223) and purified with Zymo Research RNA Clean & Concentrator-25kit (Zymo Research R1018). The DNase treated and purified total RNA (5 μg for cerebellum, neocortex, and hippocampus, or 1 μg for hypothalamus) was used for subsequent mRNA isolation and RNA-seq library preparation. The External RNA Controls Consortium (ERCC) RNA Spike-in Controls (Ambion 4456740) were diluted 1:10 (for 5 μg total RNA sample) and 1:100 (for 1 μg total RNA sample) and added to total RNA. RNA-seq libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs Inc. E7530L), with 12 PCR cycles. The qualities of the libraries were examined using Agilent High Sensitivity DNA Bioanalyzer Chips (Agilent Technologies 5067-4626) and quantified by KAPA Library Quantification Kits for Illumina sequencing platforms (KAPA Biosystems KK4824).
9
Quantification of Immune Regulators in Colon Tissue
10
Tubulin Mutant cDNA Synthesis Protocol
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Template RNA was generated from 10 ovaries with an RNA Isolation Kit (Thermo Fisher), and reverse transcription to cDNA was performed by the PrimeScript 1st strand cDNA synthesis kit (Takara, Japan). Vector (pcDNA3.1) cloning with Flag-tubulin substitution mutants (K40R) was conducted by the StarMut site-directed mutagenesis kit (GenStar, Cat#T111-01). mRNA was synthesized from linearized plasmid using the HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G(5′)ppp(5′)G (NEB), tailed with a poly(A) polymerase tailing kit (Epicentre), and purified with the RNA clean & concentrator-25 kit (Zymo Research).
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