Cell populations were sorted directly into Trizol reagent (Life Technologies). Nucleic acids were extracted according to manufacturer instructions and precipitated in isopropanol with glycogen or linear acrylamide (Ambion) used as a carrier. After 30 min of DNase I treatment (Life Technologies), RNA was purified with RNA clean and concentrator columns (Zymo). RNA integrity was verified on a Agilent 2100 Bioanalyzer Pico chip. For small-scale RNA sequencing, full-length cDNA libraries were prepared using the SMARTer Ultra Low Input RNA kit (Clontech). Amplified cDNA libraries were sheared to 200–500 bp fragments using a Covaris LE220. End repair, A-tailing and Illumina adaptor ligation reactions were carried out using a NEBNext Library Prep Kit (New England BioLabs). Libraries were sequenced on an Illumina HiSeq2500 v4 using 50-bp paired-end reads. Fastq files were aligned to mm9 using TopHat allowing 2 mismatches. Aligned features were sorted with samtools, counted with htseq-count and differential expression was determined using the ‘edgeR’ package in Bioconductor, as described55 . Microarray gene expression profiling of sorted N-mychi and N-myclo HSC and GSEA was carried out as previously described27 (link). Heat map representations of row mean centered expression values were generated using the ‘pheatmap’ package in Bioconductor.
Rna clean and concentrator column
Manufactured by Zymo Research
Sourced in United States
The RNA Clean & Concentrator columns are designed to purify and concentrate RNA samples. The columns utilize a silica-based membrane to selectively bind RNA molecules, allowing for the removal of contaminants and concentration of the target RNA. This process is achieved through a simple bind-wash-elute protocol.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Smarter ultra low input rna kit, by Takara Bio (2 mentions)
Glycogen or linear acrylamide, by Thermo Fisher Scientific (2 mentions)
Dnase 1 treatment, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer pico chip, by Agilent Technologies (2 mentions)
Hiseq 2500 v4, by Illumina (2 mentions)
Nebnext library prep kit, by New England Biolabs (2 mentions)
Le220, by Covaris (2 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (2 mentions)
Ds 11 spectrophotometer, by DeNovix (2 mentions)
Ribo zero gold, by Illumina (2 mentions)
T7 ribomax express large scale rna production system, by Promega (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Maxima h minus double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sureclean plus solution, by Meridian Bioscience (1 mentions)
Soil fecal rna kit, by Zymo Research (1 mentions)
Ribo zero rrna removal kit for bacteria, by Illumina (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Ncounter analysis system, by NanoString (1 mentions)
24 protocols using rna clean and concentrator column
1
Transcriptional Profiling of N-myc Subsets in Hematopoietic Stem Cells
2
RNA Isolation and cDNA Synthesis
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For RNA isolation 2 mL of reactor liquid samples were used. The samples were centrifuged at 12,000 rpm for 10 min. RNA extractions were carried out with the Zymo Research Soil/Fecal RNA kit (R2040, Zymo Research, Irvine, CA, United States). After lysis (bead beating), the Zymo Research kit protocol was followed. The DNA contamination was removed by Thermo Scientific Rapidout™ DNA removal kit (K2981, Thermo Fisher Scientific, Waltham, MA, United States). Ribosomal RNA was depleted using the Ribo-Zero™ rRNA Removal Kit for Bacteria (Illumina, Madison, USA) according to the manufacturer’s instructions. The rRNA depleted samples were purified via the RNA Clean and Concentrator Columns from Zymo Research (Irvine, USA). During this step, an additional in-column DNase I treatment was included to ensure complete removal of DNA. Subsequently, synthesis of double-stranded cDNA was conducted using the Maxima H Minus Double-Stranded cDNA Synthesis Kit from ThermoScientific (Waltham, USA). In the first-strand cDNA synthesis reaction, 2 μL of random hexamer primer were used. Final purification of the blunt-end double-stranded cDNA was carried out using SureClean Plus solution from Bioline (Luckenwalde, Germany). The cDNA was sequenced in the same way as the total DNA. The quality of the RNA preparation was checked by agarose gel electrophoresis (data not shown).
3
Profiling Myeloid Innate Immunity Transcripts
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Total RNA was isolated using TRIzol (Fisher) and the PureLink RNA Mini Kit (12183018A, Ambion), NEB DNase treated, and purified using RNA clean and concentrator columns (Zymo). RNA was analyzed using the mouse myeloid innate immunity panel of the NanoString nCounter Analysis System (NanoString Technologies). Raw counts of samples were normalized according to the manufacturer’s recommendations using reference genes as internal controls (Sdha, Oaz1, Rpl19, Edc3, Sap130, Hdac3, Polr2a, Ppia, Gusb, Tbp, Sf3a3, and Abcf1). Background threshold was set to the geometric mean of the negative controls. Normalization was performed using nSolver Analysis Software v4. Normalized transcript counts are shown in Figure 3—source data 1 .
4
CRISPR Guide RNA Generation Protocol
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IVT dsDNA templates were amplified by PCR from the relevant plasmid (see Supplementary Table 2 for sequences): pD1301i-SP1 was generated by cloning the 1025-1044 pSP1 spacer sequence into pD1301i (Atum Bio); the pEX-A2 family of plasmids were supplied by Eurofins; pCRISPR3 was from ref (22) ; pX330 was a gift from Feng Zhang (Addgene plasmid # 42230 ; http://n2t.net/addgene:42230 ; RRID:Addgene_42230) (23) . Primers were designed for IVT that amplified the RNA spacer and structural component and introduced the T7 promoter upstream of the gRNA sequence. IVT was performed with HiScribe T7 High Yield RNA synthesis kit (New England BioLabs) as per the manufacturer's instructions. RNAs were purified by either phenol-chloroform extraction or with RNA Clean and Concentrator Columns (Zymo Research) and eluted in DEPC treated H 2O. Synthetic crRNAs and gRNAs were supplied by Metabion and IDT, respectively (Supplementary Figure S1). gRNA concentrations were calculated by taking A 260 with a DeNovix DS-11 spectrophotometer.
5
Sorting and Isolating Mucosal-Associated Invariant T Cells
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Cells were surface stained using the flow cytometry staining protocol above. Specifically, cells were stained with propidium iodide viability stain, MR1/5-OP-RU tetramer, and antibodies to CD45, CD4, CD8. Lung samples were also stained with antibodies to CD20, CD14, and EpCAM for removal of B cells, myeloid cells, and epithelial cells. Live, CD45 + , CD4 negative, CD14/CD20/EpCAM negative were sorted into four populations: MR1/5-OP-RU + CD8+, MR1/5- OP-RU + CD8 negative, MR1/5-OP-RU-negative CD8+, and MR1/5-OP-RU-negative CD8 negative. Cells were sorted into TRIzol (Thermo-Fisher) and RNA was isolated using a Direct-zol RNA-mini kit with RNA clean and concentrator columns (Zymo Research) and treated with DNase (Qiagen).
6
EVA71 Infectious Clone Transfection
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Virus was recovered as previously described [35 (link)], following transfection of in vitro-transcribed RNA into HeLa cells. Briefly, the EVA71 infectious clone plasmid was linearized with XhoI followed by phenol–chloroform extraction. RNA was synthesized using the RiboMAX T7 express large-scale RNA production system (Promega) and purified using RNA clean and concentrator columns (Zymo Research). RNA was electroporated into HeLa cells within 0.4 mm cuvettes at 260 V for a single pulse of 25 ms using square wave. Cells were transferred to plates and incubated at 37 °C, 5 % CO2 in a humidified chamber overnight. Plates were freeze-thawed to enhance viral release from cells, and cellular debris was pelleted at 17 000 r.c.f. for 10 min. The supernatants were collected and samples titred using median tissue culture infectious dose (TCID50).
7
Generating CRISPR RNA Templates
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IVT dsDNA templates were amplified by PCR from the relevant plasmid (see Supplementary Table S2 for sequences): pD1301i-SP1 was generated by cloning the 1025–1044 pSP1 spacer sequence into pD1301i (Atum Bio); the pEX-A2 family of plasmids were supplied by Eurofins; pCRISPR3 was from (26 (link)); pX330 was a gift from Feng Zhang (Addgene plasmid # 42230; http://n2t.net/addgene:42230 ; RRID:Addgene_42230 ) (27 (link)). Primers were designed for IVT that amplified the RNA spacer and structural component and introduced the T7 promoter upstream of the gRNA sequence. IVT was performed with HiScribe T7 High Yield RNA synthesis kit (New England BioLabs) as per the manufacturer's instructions. RNAs were purified by either phenol-chloroform extraction or with RNA Clean and Concentrator Columns (Zymo Research) and eluted in DEPC treated H2O. Synthetic crRNAs and gRNAs were supplied by Metabion and IDT, respectively (Supplementary Figure S1 ). gRNA concentrations were calculated by taking A260 with a DeNovix DS-11 spectrophotometer.
8
WGS Analysis of SARS-CoV-2 in Wastewater
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Twenty-four-hour composite influent wastewater samples from the WWTPs Eindhoven and Weert, August and September 2022, were selected for WGS analysis. Nucleic acids used to determine the viral load were additionally treated with DNase and purified using RNA Clean and Concentrator columns (Zymo Research, https://www.zymoresearch.com/ ). On the purified RNA, a cDNA synthesis using random hexamer primers was performed and on the obtained cDNA a PCR was performed using the Artic V4.1 amplicon panel (ARTIC network, https://artic.network ). Libraries were prepared with the TruSeq Nano DNA kit (Illumina, https://www.illumina.com ). Paired-end short-read sequencing was performed in multiple runs a MiSeq platform (Illumina, https://www.illumina.com ) using either combinatorial-dual (CD) or unique-dual (UD) indexes (Illumina, https://www.illumina.com ).
9
HIV Drug Resistance Genotyping from DBS
10
Transcriptome Sequencing of Two Desert Rodents
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At Purdue University, total RNA was extracted from a representative portion of each sample using TRIzol® reagent (Invitrogen) according to manufacturer instructions. For the D. spectabilis and H. desmarestianus samples, ½ plate of 454 sequencing was conducted using cDNA synthesized from our initial RNA extractions with the ClonTech SMART cDNA synthesis kit [44 (link)] with a modified CDS III/3’ primer (see [43 (link), 45 (link), 46 (link)]). For Illumina sequencing, fresh RNA extractions were used to obtain total RNA for all 12 samples, which were then purified using RNA Clean and Concentrator columns (Zymo Research). RNA quality was evaluated with an Agilent 2100 Bioanalyzer before a uniquely barcoded 2 × 100 paired end library was constructed from each sample. All 12 libraries were then pooled and sequenced in two separate lanes of sequencing using an Illumina HiSeq 2000. This sequencing effort also included 12 libraries for a separate study [35 (link)], in total the same 24 libraries (12 of which were for this study) were sequenced in each lane of Illumina sequencing.
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