The mRNA was isolated from putative transgenic using the PolyATract® mRNA isolation system III (Promega). RT-PCR amplification was performed in a total volume of 20 μl containing 1X OneStep RT-PCR buffer (Qiagen), dNTPs 10 mM, 5X Q Solution (Qiagen), 1.0 μM of each primer (PAT-F and PAT-R), 1.0-unit Reverse Transcriptase enzyme mix (Qiagen) and 300 ng of mRNA. For reverse transcription reaction, the mix was incubated at 55°C at 30 min followed by initial PCR activation at 95°C for 15 min. The touch-down PCR was performed as described in the PCR section above. RT-PCR was also done to amplify the CYP79A1 antisense cDNA product whose sequence was found to be identical to the corresponding reverse sequence of a native CYP79A1 fragment.
Onestep rt pcr buffer
Manufactured by Qiagen
Sourced in Germany, United States
The OneStep RT-PCR Buffer is a reagent used in the process of reverse transcription and polymerase chain reaction (RT-PCR) for the detection and analysis of RNA targets. It facilitates the conversion of RNA to complementary DNA (cDNA) and the subsequent amplification of the cDNA sequence.
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Lab products found in correlation
Onestep rt pcr kit, by Qiagen (20 mentions)
Onestep rt pcr enzyme mix, by Qiagen (14 mentions)
Q solution, by Qiagen (2 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
One step enzyme mix, by Qiagen (2 mentions)
Facsaria fusion high speed cell sorter, by BD (2 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (2 mentions)
One step rt pcr enzyme, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Polyatract mrna isolation system 3, by Promega (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Rnasin, by Promega (1 mentions)
Geldoc system, by Clinx (1 mentions)
Pcr buffer, by Qiagen (1 mentions)
Taq dna polymerase, by Qiagen (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Mtb lysate, by BEI Resources (1 mentions)
Facsdiva software, by BD (1 mentions)
37 protocols using onestep rt pcr buffer
1
mRNA Isolation and RT-PCR Amplification
2
RT-PCR for Variant Identification
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The RT-PCR primers were designed using the automated Primer3 [41 (link)]. The RT-PCR was performed in 25 μl reaction containing final concentration (1x) of 5μl of 5x Qiagen One-step RT-PCR buffer, 400 μM of each dNTPs, 0.6 μM each of forward and reverse primers, 1μl of Qiagen One step RT-PCR enzyme mix and template RNA up to 400ng and volume made up to 25 μl using PCR grade water. The cDNA of about 200ng was PCR amplified for 30 min at 54° C, initial heat activation for 15 min at 95° C, 33 cycles of initial denaturation for 30 sec at 94° C, annealing for 30 sec at 61° C, extension for 30 seconds at 72° C, final extension for 5 min at 72° C. The forward and reverse primers used for RT-PCR and their amplicon sizes are mentioned in (Supplementary Table S1 ). To identify each of the variant, variant specific primers that span exon junctions were designed. All primers are listed in the Supplementary Table S1 .
3
TCR Sequencing of M. tuberculosis-Specific CD8+ T Cells
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Single-cell TCR sequencing was performed as described previously (36 (link), 37 (link)). Briefly, cryopreserved PBMCs from 12 healthy adolescents with evidence of M. tuberculosis infection (determined by QuantiFERON-Plus and/or TST positive test results) were thawed, rested for 6 h, and stimulated for 12 h with M. tuberculosis lysate (10 μg/ml; BEI Resources) in the presence of anti-CD49d Ab (1 μg/ml), and anti-CD154-PE (10 μl/ml). Cells were stained with LIVE/DEAD Fixable Aqua Stain (Thermo Fisher Scientific) and then with Abs (Supplemental Table I ). Single, activated (i.e., CD69+CD137+ and/or CD69+CD154+) TCRαβ+ CD8+ cells were sorted by FACS (BD FACSAria II) into 96-well plates containing OneStep RT-PCR buffer (QIAGEN). TCRαβ sequences were amplified using a panel of TCRαβ primers and further amplified in a nested PCR before sequencing on a MiSeq (Illumina) instrument, as described previously (36 (link), 37 (link)). TCR sequences from six of these 12 adolescents were published recently (38 (link)), as indicated in Supplemental Table II .
4
RT-PCR for Influenza Virus Subtyping
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RT-PCR for the classification of the influenza virus subtypes was performed as described previously [11 (link)] with minor modifications. Briefly, viral RNA was extracted from sample specimens using a QIAamp® Viral RNA Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed with a OneStep RT-PCR kit (QIAGEN) using individual primer sets for H1N1dpm, H1N1 seasonal influenza before 2009, or H3 (Supplementary Table S1 ). The reaction components for RT-PCR were prepared by mixing 2 μL of 5× QIAGEN OneStep RT-PCR Buffer, 0.4 μL of dNTP mix, 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), 0.32 μL of QIAGEN OneStep RT-PCR Enzyme Mix, 5.68 μL of Nuclease-free H2O, and 1.2 μL of Viral RNA per reaction. The PCR conditions were 50 °C for 30 min and 94 °C for 3 min, followed by 45 cycles of 94 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 7 min. The PCR products were subjected to electrophoresis on a 2% agarose gel. RNA extracted from each positive control virus was used as the standard RNA. The concentration of each sample was calculated by fitting the infection titer of each virus to create a standard curve.
5
Multiplexed αβ-TCR Sequencing Protocol
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Multiplexed αβ-TCR sequencing was done following previously established protocols (33 (link)). In brief, single spheromer+ CD8+ T cells (for influenza-M1, HCMV-pp65, and SARS-CoV-2 specificities) were sorted into 96-well plates containing 12 μl of OneStep RT-PCR buffer (Qiagen). Reverse transcription was done using the OneStep RT-PCR kit (Qiagen), and the resulting complementary DNA (cDNA) was used for TCRα and TCRβ amplification using multiplex primers. DNA barcodes were also incorporated within the amplified sequences before processing the samples in a single MiSeq2 × 300–base pair sequencing run. The paired sequencing reads were joined, demultiplexed, and mapped to the human TCR reference dataset available at the international ImMunoGeneTics information system as reported previously (33 (link)).
6
Amplification and Sequencing of RVH NSP5
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To amplify the NSP5 segment of Rotavirus H (RVH), specific primers were designed using Primer3Plus [69 (link)] with manual inspection in Clonemanager 9 (Sci-Ed Software, Cary, North Carolina, USA). As a negative control nuclease free water was used. The following primers were used: forward 5’-GGAACTAAAAACTTCAATCGTTGCTG-3’ and reverse 5’-GTTTTTATTGATGACCTCAGGGGC-3’ . Before amplification, an initial denaturation step with 10 μl of extracted RNA for 5 min at 97°C was performed. To the denatured RNA 40 μl of the PCR mix containing 10 μl of One Step RT-PCR Buffer (5X; Qiagen, Hombrechtikon Switzerland), 1.6 μl forward Primer (100 μM) and 1.6 μl reverse Primer (100 μM), 2 μl dNTP Mix (10 mM each), 2.0 μl One Step Enzyme Mix (Qiagen Hombrechtikon, Switzerland), 22.6 μl nuclease free water, and 0.2 μl RNasin (Promega, Madison, Wisconsin, USA) was added. The PCR conditions were as follows: 30 min at 50°C, 15 min at 95°C, 40 cycles of 50 sec at 94°C, 50 sec at 55°C and 60 sec at 72°C, a final extension step for 10 min at 72°C. The PCR product was analyzed by agarose gel electrophoresis. The expected band of approx. 666 bp was cut out with a scalpel blade, and the DNA was extracted and sequenced as described above.
7
DNA and RNA Quality Evaluation by PCR
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We performed end-point PCR to determine the quality of the isolated DNA and end-point RT-PCR to determine the quality of the extracted RNA [6 (link),16 ]. The PCR cycling conditions consisted of an initial denaturation step at 95 °C for 15 min; 40 cycles at 95 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s; and a final extension step at 72 °C for 7 min. In each reaction, a 5 μL aliquot of DNA was amplified in a total volume of 25 μL containing PCR buffer (10X, Qiagen), 2.5 mM MgCl2, 0.25 mM deoxynucleotide triphosphate, 25 pM of each primer, one unit of Taq DNA polymerase (Qiagen) and deionized (DI) water. For the RNA assays, 5 μL of isolated RNA was amplified in a total volume of 25 μL containing One-step RT-PCR buffer (5X, Qiagen), 0.25 mM deoxynucleotide triphosphate, 25 pM of each primer, 1 μL of RT-PCR One-step enzyme mix (Qiagen) and DI water. The following thermal profile was used for RT-PCR: 30 min reverse transcription at 50 °C; 15 min pre-denaturation at 95 °C; 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C; and a final extension at 72 °C for 10 min. The resulting PCR and RT-PCR products were separated on 2% agarose gels containing ethidium bromide (EtBr) and imaged with a Gel-Doc System (Clinx Science Instruments, Shanghai, China).
8
Single-Cell Sequencing of Tumor-Infiltrating Lymphocytes
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Cells were thawed and stained with multicolor panels (Suppl. Table 1). Antibodies were used according to the manufacturer’s instructions. TILs and TUM samples from each patient were processed in parallel to minimize instrument and staining variability. For single cell sequencing, single cells were index-sorted directly into 96-well plates pre-filled with OneStep RT-PCR buffer (Qiagen) as previously described.24 For TCRβ repertoire sequencing, bulk cells were FACS-sorted into tubes prefilled with RPMI1640 containing 2% FBS. DNA was isolated immediately after sorting using the DNeasy Blood & Tissue Kit (Qiagen) and stored at 4°C until further processing. All cells were sorted using a FACSAria™ Fusion high-speed cell sorter (BD Biosciences) equipped with a 70 µm nozzle.
9
Single-cell TCR sequencing of M.tb-specific CD8+ T cells
10
Single-Cell Immune Phenotyping and TCR Sequencing
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Lymph node cell suspensions were stained with multi-parameter antibody panels (Suppl. Tab. 1) according to the manufacturers’ instructions. Single cell index sorting into 96-well plates pre-filled with OneStep RT-PCR buffer (Qiagen) for T cell receptor (TCR) αβ and phenotype sequencing was done as described previously [7 (link)]. Index sorting guaranteed highly accurate 13-dimensional immune phenotyping of every single sorted cell at the protein level with accurately assigned immune phenotypes in > 99% of sorted T cells [7 (link)]. All cells were sorted using a FACSAria™ Fusion high-speed cell sorter (BD Biosciences) equipped with a 70 µm nozzle and were frozen at -80 ºC immediately after sorting until further processing. Multi-parameter immune phenotyping was performed on all ten patients in the study; sufficient material for single cell index sorting was available from nine out of ten patients. Index sorting data were exported from FACSDiva software (BD Biosciences) as “comma-separated values” (.csv) files according to the manufacturer’s instructions and combined with single cell sequencing data.
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