WBCs were cryopreserved in CryoStor CS10 Freezing Media (#210102) at ~10×106 cells/mL. WBCs were thawed quickly at 37°C and immediately spiked into 9 mL of complete RPMI media then centrifuged at 500 g for 5 min and media removed. Cells were resuspended in 600 μL RLT Plus (QIAGEN) and Beta-mercaptoethanol (BME) lysis buffer (10 μL BME:1 mL RLT Plus). RNA was extracted using the AllPrep DNA/RNA/Protein Mini Kit according to manufacturer’s instructions (QIAGEN, 80004). RNA quality was determined by Agilent 2200 TapeStation and quantified by Qubit (Life Technologies). Samples with RINs of R 6.8 and a minimum of 1 μg were used to prepare next-generation sequencing libraries with the Illumina TruSeq Stranded mRNA Library Prep Kit and sequenced with an Illumina NovaSeq 6000 instrument. Analysis of library complexity and high per-base sequence quality across all reads was performed using FastQC software. Other bioinformatic steps included trimming of low-quality bases, short reads, and adaptor sequences, with the fastqc-mcf. tool; removal of mycoplasma, mitochondria, and rRNA contaminant sequences with FASTQ Screen; read alignment to GRCh37/hg19 using TopHat2; filtering of high-quality mapped reads with SAMtools; and final quality performed using RSeQC. Gene level counts were obtained using HTSeq (Anders et al., 2015 (link)).
Rlt plus
Manufactured by Qiagen
Sourced in Germany, United States
The RLT Plus is a lysis buffer designed for the efficient lysis and stabilization of RNA from a variety of sample types. It is a component of Qiagen's RNA extraction and purification solutions, and is used to disrupt cells and tissues and release RNA while inhibiting RNase activity.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy plus mini kit, by Qiagen (6 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Nextseq 500 sequencer, by Illumina (4 mentions)
Taqman probe, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Allprep universal kit, by Qiagen (3 mentions)
Truseq stranded mrna library prep kit, by Illumina (3 mentions)
Allprep dna rna protein mini kit, by Qiagen (3 mentions)
Micro rna isolation kit, by Qiagen (3 mentions)
Nextseq sequencer, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Quick rna miniprep kit, by Zymo Research (3 mentions)
Total rna nano chip, by Agilent Technologies (3 mentions)
Clc genomics workbench, by Qiagen (3 mentions)
2 mercaptoethanol, by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Qiashredder column, by Qiagen (2 mentions)
Rna 6000 nano kit, by Agilent Technologies (2 mentions)
2 beta mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
58 protocols using rlt plus
1
Cryopreserved WBC RNA Sequencing
2
Transcriptomic Analysis of T21 and D21 WBCs
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Peripheral blood was collected in EDTA vacutainer tubes (BD, 366643) from ten individuals with T21 and nine D21 controls. Blood was centrifuged at 500 × g for 15 min to separate plasma, buffy coat, and red blood cells. WBCs were isolated from the buffy coat fraction by RBC lysis and washed with our sorting buffer (see below) according to the manufacturer’s instructions (BD, 555899). WBCs were cryopreserved in CryoStor CS10 Freezing Media (210102) at ~10 million cells/mL. WBCs were quickly thawed at 37 °C and immediately spiked into 9 mL of sorting media and centrifuged at 500 × g for 5 min and the media removed. Cells were lysed in 600 µL RLT plus (Qiagen) and β-mercaptoethanol (BME) lysis buffer (10 µL BME:1 mL RLT plus). RNA was extracted using the AllPrep DNA/RNA/Protein Mini Kit according to the manufacturer’s instructions (Qiagen, 80004). RNA quality was determined by Agilent 2200 TapeStation and quantified by Qubit (Life Technologies). Samples with RIN of 6.8 or greater and a minimum of 1 µg were sent to Novogene for stranded library prep and 2 × 150 paired-end sequencing.
3
Comparative Embryonic Profiling
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Reciprocal natural timed matings were set up between C57BL6/Babr and CAST/EiJ animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic days 6.5 (E6.5) and 7.5 (E7.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3+ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) [9 (link)] and CAST males (denoted as matDKO/CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N = 4) and ExE (N = 8) samples were pooled (an estimated ~ 2500 cells), washed in PBS, and then flash-frozen in 10 μL of nuclear lysis buffer (Sigma-Aldrich). Single E7.5 epiblast and ExE samples were individually frozen in 10 μL of buffer RLT Plus (Qiagen).
4
Isolation of High-Quality RNA from J-Lat and CD4+ T Cells
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For RNA isolated from J-Lat 10.6 cells, cells first were passaged and split equally three times prior to isolation. Either J-Lat 10.6 or wild type for CCNT1 or knocked out for CCNT1 were each treated with TNFα (Peprotech, 300-01A) at 10 ng/mL or unstimulated in triplicate. For primary cell experiments, knockouts were performed similar to the “Primary CD4+ T cell activation Test,” and RNA was isolated after LRA treatment. In both J-Lat and primary CD4+ T cell isolation experiments, 0.1–2 × 106 cells were isolated and resuspended in 350 μL of RLT Plus (Qiagen, 1053393) + 1% 2-mercaptoethanol (Millipore Sigma, M3148). Cells were frozen in buffer RLT Plus until RNA isolation. Thawed RLT lysates were then run over a QIAshredder column (Qiagen, 79654) and subsequently a gDNA eliminator column. A Qiagen RNeasy Plus Mini Kit was then used in order to obtain purified total RNA. RNA was submitted for TapeStation RNA assay or HighSense RNA assay (Fred Hutch Core Facilities Seattle, Washington, DC, USA), and RINe scores were all found to be ≥9.6.
5
Transcriptome Analysis of E2-Induced Cells
6
Quantifying mRNA Expression after β-AR Modulation
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For mRNA analysis, cells were preincubated with β-AR antagonists for 30 minutes before incubation with β-AR agonists for 2 hours in 24-well plates. Cells were then washed with 1X phosphate-buffered saline (PBS; Sigma), lysed in RLT plus (a guanidine-rich buffer; Qiagen), and frozen at −20°C. Messenger RNA was extracted by using RNeasy Plus Mini Kit (Qiagen). The cDNA was synthesized by using Sprint RT Complete-Double PrePrimed (Clontech, Mountain View, CA, USA). Cytokine mRNAs were measured by quantitative PCR (Eppendorf, Hauppauge, NY, USA) and normalized to the housekeeping gene RpL13A by generating a DCt value. Primer sequences can be found in the following references or Table 2 .18 (link),24 (link) Fold values were generated by normalizing to the vehicle control. Vehicle control samples were used to assay for baseline levels of β-AR.
7
Isolation and Analysis of Circulating Plasmacytoid Dendritic Cells
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Blood was collected in Lithium–Heparin tubes (BD Vacutainer, Franklin Lakes, NJ, USA). Circulating pDCs were isolated from peripheral blood by positive selection using CD304 (BDCA-4) magnetic beads on AutoMacs Pro (Milteny Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. PDCs were lysed in RLTplus (Qiagen, Hilden, Germany) with b-mercaptoethanol. RNA from pDCs was isolated using the Allprep Universal Kit (Qiagen), according to the manufacturer’s instructions. RNA quantification was performed on Qubit 2.0 fluorimeter (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) using the Qubit RNA HS Assay Kit (Invitrogen). RNA integrity (RNA integrity number ≥8.0) was assessed using RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA).
8
Sample Collection and Processing
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(Figure 1 ) consisted of cutting off the distal swab tip (~0.5 cm) with clean scissors immediately after collection, placing the tip into a cryovial with 350μL of RLT Plus® (Qiagen) with 350 μL 2-beta mercaptoethanol (Invitrogen), and vortexing the cryovial for 30 seconds, and transporting the sample to the laboratory on ice for storage at −80° C. The proximal part of the swab was placed in ESwab™ liquid Amies and processed as previously described.
9
Milk Fractionation and Cell Isolation
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Milk was separated into the lipid layer, de-fatted (“skim”) milk, and cellular portion by centrifugation at 710×g for 20 min. The cell pellet was washed three times prior to staining for flow cytometry or freezing in RLT plus (Qiagen) for subsequent viral quantification.
10
Simultaneous DNA and RNA Extraction with qPCR
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