RNA preparation and sequencing was performed as described previously [42 (link)]. Briefly, approximately 100 mg of plant material (5 leaf discs) was used for RNA extraction using the Qiagen RNeasy Plant Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol, with an added DNase treatment interruption step. The DNase treatment was performed on column using the Invitrogen PureLink™ DNase set (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s protocol. The RNA quality and concentration were both corroborated by ND-1000 NanoDrop and by a 2100 Bioanalyzer using RNA Nano chips (Agilent Technologies, CA, USA). Polyadenylated messenger RNA was captured from 200 ng total RNA per sample using magnetic beads and Illumina adaptors with sample-specific barcode sequences that were ligated before subsequent library amplification using PCR via the Illumina TruSeq RNA poly-A selection kit. Sequencing of 150 bp paired-end libraries was carried out using the Illumina NovaSeq6000 S4 platform (SciLifeLab, Stockholm, Sweden). All raw sequencing data in this study have been deposited in National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA755645.
Purelink dnase set
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia
The PureLink™ DNase Set is a laboratory equipment product designed for the removal of DNA contaminants from RNA samples. It provides a simple and efficient way to eliminate DNA interference during downstream RNA-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Purelink rna mini kit, by Thermo Fisher Scientific (62 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (12 mentions)
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (7 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (6 mentions)
2100 bioanalyzer, by Agilent Technologies (6 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (6 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (3 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (3 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Itaq universal sybr green supermix, by Bio-Rad (3 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
68 protocols using purelink dnase set
1
RNA Extraction and Sequencing from Plant Material
2
Brain Tissue RNA Isolation and qPCR Analysis
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Weighed brain tissue samples were homogenized in ultra-pure-guanidine isothiocyanate (Sigma-Aldrich, Cat# 50,983) using innuSPEED Lysis Tubes P in SpeedMill Plus (Analytik Jena, Germany, Cat# 845-CS-1020250). RNA was isolated using Pure Link RNA mini kit (Invitrogen, Cat# 12183018A) as recommended by the manufacturer. DNA contamination was removed using PureLink™ DNase Set (Invitrogen, Cat# 10,977,035). RNA purity and the concentrations were determined using a NanoDrop spectrophotometer (Epoch, BioTek) and total RNA was converted into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat# 4,368,814) according to supplier’s protocol. Quantitative PCR (qPCR) was performed using with the primers shown at Supplementary Table 1. AMPIGENE qPCR Green Mix Hi-ROX (Enzo Life Sciences, Cat# ENZ-NUC104) was used under the following conditions: 95 °C for 2 min for initial denaturation, followed by 5 s (40 cycles) at 95 °C and 30 s at 57 °C. Data were generated in the final step at 95 °C for 15 s and melting curves (65 to 95 °C) were acquired at the end for each primer set. Relative gene expression was calculated by 2−ΔΔCt method by normalizing gene expression to β-actin (Aktas et al. 2015 (link)). Data were generated for four mice per group and each biological replicate has four technical replicates.
3
Vascular Smooth Muscle Cell Analysis
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PureLink DNAse set and PureLink RNA mini kit were purchased from Invitrogen (Grand Island, NY, USA). High-Capacity cDNA Reverse Transcription kit and Power SYBR green Mastermix were purchased from Applied Biosystems (Foster City, CA, USA). Polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad (Berkeley, CA, USA). Amersham ECL Plus Western Blotting Detection reagents were purchased from GE Healthcare (Rockford, IL, USA). Xuesaitong injection (containing 400 mg PNS per ampule) was obtained from the Kunming Pharmaceutical Group Co., Ltd. (Yun Nan province, China). Rabbit anti-Notch3, mouse anti-SM22α, and rabbit anti-OPN were purchased from Abcam (Cambridge, MA, USA). TRITR-phalloidin, FITC-phalloidin, and mouse anti-α-SM-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-β-actin and mouse anti-β-tubulin were purchased from Sigma (St. Louis, MO, USA).
4
RNA Extraction from Cell Lines
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HCT116, SW620, Caco-2, HT-29, and CCD 841 CoN cells were cultured until 70% confluency. The cells were then trypsinized (Corning™, Catalog #: 25-053-CI) and subsequently spun down into cell pellets. RNA was then extracted from the cell pellets using the Invitrogen™ PureLink™ RNA Mini Kit (Invitrogen™, Carlsbad, CA, USA, Catalog #: 12183018A) as per the manufacturer’s protocol. The cell lysates were homogenized by being passed five times through 21-gauge tuberculin syringes (BD™, Franklin Lakes, NJ, USA, Catalog #: 309624) as per the manufacturer’s (Invitrogen™) recommendation. On-column DNase I digestion was also performed using the Invitrogen™ PureLink™ DNase Set (Invitrogen™, Catalog #: 12185010) as per the manufacturer’s protocol. The purified RNA was then placed into single-use aliquots and stored at −80 °C.
5
Osteogenic Gene Expression in HDPCs
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HDPCs were treated with odontogenic medium containing ASA for 14 d, and then, the TRIzol reagent (Invitrogen; Thermo Fisher Scientific) was applied to lyse the cells for RNA extraction and purification using PureLink™ RNA Mini Kit incorporating with PureLink™ DNase Set (Invitrogen) according to the manufacturer's instructions. RNA yield and RNA quality were determined by using NanoDrop™ 2000 UV Visible Spectrophotometer (Thermo Fisher Scientific). The extracted RNA of 0.2 μ·g from each sample was utilized in one-step RT-qPCR using SuperScript™ III Platinum™ One-Step qRT-PCR Kit (Invitrogen) on StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific). The target TaqMan probes of dentin sialophosphoprotein (DSPP; Hs00171962_m1; Thermo Fisher Scientific) and RUNX2 (Hs00231692_m1; Thermo Fisher Scientific) were applied. The housekeeping gene, β-actin (Hs03023943_g1; Thermo Fisher Scientific), was used to normalize gene expression levels of each target gene, and the 2−ΔΔCt calculation was conducted for the relative differences in the gene expressions (n = 6). PCR was set under standard cycling conditions as described: 50°C for 15 min followed by 95°C for 2 min, then 40 cycles of 95°C for 15 s, and 60°C for 30 s.
6
RNA Isolation from Mouse Colonic Tissue and DRG
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RNA from mouse colonic tissue was isolated using the PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA) and from mouse DRG using the PureLink RNA Micro Kit (Invitrogen) according to the manufacturer's instructions without modifications as described previously.13 (link),15 ,18 (link) All samples underwent an on-column DNase treatment with the PureLink DNase Set (Invitrogen). The elution volumes were 60 µL for mouse colonic tissue RNA and 20 μL for mouse DRG RNA. RNA was aliquoted and stored at −80°C until use. RNA quality was assessed using the 2100 Bioanalyzer Instrument (Agilent Technologies, Inc, Santa Clara, CA) or the 2200 TapeStation System (Agilent Technologies, Inc). All samples had an RNA integrity number of 6.5 or higher.
7
PNA-Based Bacterial Detection Protocol
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All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received unless otherwise noted. Carboxylic acid-functionalized, 3-µm-diameter polystyrene microspheres were purchased from Polysciences, Inc. (Warrington, PA, USA). Peptide nucleic acid (PNA) was purchased from Bio-Synthesis, Inc. (Lewisville, TX, USA) as HPLC-purified and lyophilized powders. The PNA probe for detecting E. coli 16S rRNA was NH2-(CH2CH2O)12- CTC CTT CCC TCA TTT CA [27 (link)]. For positive control experiments, the universal PNA probe NH2-(CH2CH2O)12- CTG CCT CCC GTA GGA was used [31 (link)]. Methoxy-polyethylene glycol amine, CH3O-(CH2CH2O)3-NH2 (MW 350) was obtained from Nanocs, Inc. (New York, NY, USA). Pre-pulled borosilicate micropipettes with 2 µm inside tip diameter were purchased from World Precision Instruments, Inc. (Sarasota, FL, USA). All bacteria: E. coli (ATCC 25922), Pseudomonas fluorescens (ATCC 13525), and Pseudomonas putida (ATCC 12633), and culture medium ingredients, soy agar and nutrient agar were purchased from ATCC, Inc. (Manassas, VA, USA). PureLink RNA Mini Kit, PureLink DNase Set, RNase free water, RNase-free pipette tips, RNase-away reagent and RNase-free microfuge tubes were purchased from Invitrogen Life Technologies (Grand Island, NY, USA).
8
Quantifying Bacterial Conjugation and Phage Delivery
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Bacteria samples
were collected by centrifugation after 1 h of conjugation and phage
delivery assays. Total bacterial RNA was extracted from the pellets
using a PureLink RNA mini kit (Invitrogen, USA) with on-column DNase
treatment (PureLink DNase Set, Invitrogen, USA) to remove residual
DNA. The quality and quantity of RNA were determined by using a NanoDrop
1000 spectrophotometer (Thermo Scientific, USA). RNA was transcribed
to cDNA by reverse transcription PCR (RT-PCR) using a High-Capacity
RNA-to-cDNA kit (Invitrogen, USA).
qPCR was performed to quantify
the transcribed cDNA of conjugation-related genes (traI, traJ, and trbAp) and phage delivery-related
genes (lamB and malT) (Table 2 ). 16S rRNA gene was
included as a reference gene.86 (link) Differential
gene expression relative to the 16S rRNA gene was quantified by the
2–ΔΔCT method,87 (link) and the cycle threshold (CT) values used were the means of independent
triplicates. Heatmap presenting the overall RT-PCR and qPCR data of
targeted genes was performed in Origin Pro 2021. Details of the RT-PCR
and qPCR protocols are available in theSupporting Information (Text S3).
were collected by centrifugation after 1 h of conjugation and phage
delivery assays. Total bacterial RNA was extracted from the pellets
using a PureLink RNA mini kit (Invitrogen, USA) with on-column DNase
treatment (PureLink DNase Set, Invitrogen, USA) to remove residual
DNA. The quality and quantity of RNA were determined by using a NanoDrop
1000 spectrophotometer (Thermo Scientific, USA). RNA was transcribed
to cDNA by reverse transcription PCR (RT-PCR) using a High-Capacity
RNA-to-cDNA kit (Invitrogen, USA).
qPCR was performed to quantify
the transcribed cDNA of conjugation-related genes (traI, traJ, and trbAp) and phage delivery-related
genes (lamB and malT) (
included as a reference gene.86 (link) Differential
gene expression relative to the 16S rRNA gene was quantified by the
2–ΔΔCT method,87 (link) and the cycle threshold (CT) values used were the means of independent
triplicates. Heatmap presenting the overall RT-PCR and qPCR data of
targeted genes was performed in Origin Pro 2021. Details of the RT-PCR
and qPCR protocols are available in the
9
Microglia RNA Isolation and Analysis
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RNA was extracted from samples of FACS‐sorted microglia with PicoPure™ RNA Isolation Kit (#KIT0204, Thermo Fisher Scientific) following manufacturer instructions with minor modifications. RNA was precipitated with 70% ethanol. To avoid genomic DNA contamination a DNAse step was performed using Invitrogen™ PureLink™ DNase Set (#12185010, Invitrogen). RNA quantity and purity were assessed with the Pico Kit Assay on the Agilent 2100 Bioanalyzer System.
10
Isolation and Characterization of RNA from Goat MSC and Blood
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Total RNA was isolated from MSC using a Pure Link RNA Mini Kit (Ambion, USA) according to the manufacturer’s instructions. The isolation was supported by a DNAse digestion to remove genomic DNA using a PureLinkDNase Set (Invitrogen, USA). The total RNA from the blood samples was isolated using a NucleoSpin RNA Blood kit (Macherey-Nagel, Germany). Erythrocytes were degraded with the use of Red Blood Cell Lysis Buffer (Roche, Switzerland). The isolated RNA has been subjected to a qualitative and quantitative assessment using a NanoDrop spectrophotometer (NanoDrop, USA) and Bioanalyzer 2100 (Agilent Technologies, France). Information about the mean values of RNA is shown in Table 1 .
Mean values of RNA extracted from the MSC and whole blood of goats
| Mean values | MSCa | Blood |
|---|---|---|
Quantity [ng/μL] | 167.88 | 69.41 |
A260/A280 ratio | 1.98 | 1.82 |
A260/A230 ratio | 1.44 | 1.20 |
| RIN# | 5.25 | 6.0 |
aMSC – milk somatic cells; #RIN – RNA Integrity Number
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