Cells were lysed in RLT buffer (QIAGEN) after sorting (ex vivo) and/or after 24 h of in vitro activation. The RNA extraction was performed with the RNeasy micro kit (QIAGEN) according to the manufacturer’s instructions. Reverse transcription was performed on total RNA using superscript II polymerase (Invitrogen) with random hexamers and oligo deoxythymine and deoxynucleotide triphosphates (Promega). Transcript quantification was done by real-time PCR on a 480 LightCycler instrument (Roche) using a master mix (Eurogentec), and the following TaqMan Assays (Life Technologies): TLR7 (Hs00152971_m1), TLR8 (Hs00152972_m1), ITGB8 (Hs00174456_m1), INHBA (Hs01081598_m1), IL12A (Hs01073447_m1), IL12B (Hs01011518_m1), TGFB1 (Hs00998133_m1), and TNFSF13B (Hs00198106_m1). The second derivative maximum method was used to get the Crossing points from each analyte. The relative expression of transcripts was quantified by comparison to the mean of the two housekeeping genes: HPRT1 (Hs02800695_m1) and GAPDH (Hs99999905_m1).
Superscript 2 polymerase
Manufactured by Thermo Fisher Scientific
Sourced in Japan
Superscript II polymerase is a reverse transcriptase enzyme used for the synthesis of first-strand cDNA from RNA templates. It exhibits high-temperature stability and reduced RNase H activity, enabling efficient cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 instrument, by Roche (4 mentions)
Rlt buffer, by Qiagen (3 mentions)
Rnaeasy micro kit, by Qiagen (3 mentions)
Taqman assay, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Icycler thermocycler, by Bio-Rad (1 mentions)
Oligo dt, by Thermo Fisher Scientific (1 mentions)
Taqman primer probes sets, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Sera mag oligo dt beads, by Thermo Fisher Scientific (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Trizol protocol, by Merck Group (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Oligo dt, by Promega (1 mentions)
Dntps, by Promega (1 mentions)
Nanodrop nd 1000, by Agilent Technologies (1 mentions)
Rneasy kit, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
13 protocols using superscript 2 polymerase
1
Quantifying Immune Transcripts by RT-qPCR
2
Comprehensive Gene Expression Profiling
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Cells were sorted and lysed in RLT buffer. RNA extraction was performed using the RNAeasy micro kit (QIAGEN) according to manufacturer’s instructions. Total RNA was retrotranscribed using the superscript II polymerase (Invitrogen) in combination with random hexamers, oligo dT, and dNTPs (Promega).
Transcripts were quantified by real time PCR on a 480 LightCycler instrument (Roche). Reactions were performed in 10 µl, using a master mix (Eurogentec), with the following TaqMan Assays (all from Life Technologies): BCL6 (Hs00153368_m1), PRMD1 (Hs00153357_m1), BTLA (Hs00699198_m1), CXCR4 (Hs00607978_s1), CXCR5 (Hs00540548_s1), CXCL13 (Hs00757930_m1), ICOS (Hs00359999_m1), IL-21 (Hs00222327_m1), PDCD1 (Hs01550088_m1), SH2D1A (Hs00158978_m1), CCR7 (HS 00171054_m1), CD200 (Hs01033303_m1), IL-4 (Hs00174122_m1), TNF (Hs00174128_m1), MAF (Hs00193519_m1), GATA-3 (Hs00231122_m1), TBX-21 (Hs00203436_m1), RORC (Hs01076112_m1), FOXP3 (Hs00203958_m1), IL-5 (Hs00174200_m1), IL-13 (Hs99999038_m1), IFNG (Hs00174143_m1), and IL-17A (Hs00174383_m1). Crossing points (Cp) from each analyte were obtained using the second derivative maximum method, and the transcripts were quantified as fold changes in comparison to the mean of the two housekeeping genes (B2M [Hs99999907_m1] and RPL34 [Hs00241560_m1]).
Transcripts were quantified by real time PCR on a 480 LightCycler instrument (Roche). Reactions were performed in 10 µl, using a master mix (Eurogentec), with the following TaqMan Assays (all from Life Technologies): BCL6 (Hs00153368_m1), PRMD1 (Hs00153357_m1), BTLA (Hs00699198_m1), CXCR4 (Hs00607978_s1), CXCR5 (Hs00540548_s1), CXCL13 (Hs00757930_m1), ICOS (Hs00359999_m1), IL-21 (Hs00222327_m1), PDCD1 (Hs01550088_m1), SH2D1A (Hs00158978_m1), CCR7 (HS 00171054_m1), CD200 (Hs01033303_m1), IL-4 (Hs00174122_m1), TNF (Hs00174128_m1), MAF (Hs00193519_m1), GATA-3 (Hs00231122_m1), TBX-21 (Hs00203436_m1), RORC (Hs01076112_m1), FOXP3 (Hs00203958_m1), IL-5 (Hs00174200_m1), IL-13 (Hs99999038_m1), IFNG (Hs00174143_m1), and IL-17A (Hs00174383_m1). Crossing points (Cp) from each analyte were obtained using the second derivative maximum method, and the transcripts were quantified as fold changes in comparison to the mean of the two housekeeping genes (B2M [Hs99999907_m1] and RPL34 [Hs00241560_m1]).
3
Quantitative Analysis of Cholesterol and Hedgehog Signaling
4
Pachytene Spermatocyte RNA-seq Analysis
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RNA isolation. Next generation sequencing of mRNA was performed on elutriated adult pachytene Ddx4:Cre;Dcr1fx/fx mutant and control spermatocytes according to Illumina's protocol using 200 ng of RNA. Total RNA was isolated using the Trizol protocol (Sigma-Aldrich). Genomic DNA was removed by DNase treatment. RNA integrity was measured using an Agilent 2100 bioanalyzer and samples had RIN >7.0.
RNA-seq library preparation. 200 ng of RNA was used to isolate polyA + RNA using Sera-Mag oligo(dT) beads (Thermo Scientific), fragmented with an NEBNext kit (BioLabs). First-strand cDNA was synthesized with random primers using the Superscript II polymerase (Invitrogen). Second strand cDNA, end-repair, A-base addition, and ligation of the Illumina PCR adaptators were performed according to Illumina's protocol. Libraries were then selected for 100 bp cDNA fragments on a 3.5% agarose gel and PCR-amplified using Phusion DNA polymerase (Finnzymes) for 15–18 cycles. PCR products were then purified on a 2% agarose gel and gel-extracted. Each library was quality controlled (product size and concentration) by an Agilent 2100 bioanalyzer. Single-end libraries were sequenced as 100-mers on a Genome Analyzer II flowcell according to Illumina's protocol at a depth of ∼16.2–18.0 million reads per library.
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RNA isolation. Next generation sequencing of mRNA was performed on elutriated adult pachytene Ddx4:Cre;Dcr1fx/fx mutant and control spermatocytes according to Illumina's protocol using 200 ng of RNA. Total RNA was isolated using the Trizol protocol (Sigma-Aldrich). Genomic DNA was removed by DNase treatment. RNA integrity was measured using an Agilent 2100 bioanalyzer and samples had RIN >7.0.
RNA-seq library preparation. 200 ng of RNA was used to isolate polyA + RNA using Sera-Mag oligo(dT) beads (Thermo Scientific), fragmented with an NEBNext kit (BioLabs). First-strand cDNA was synthesized with random primers using the Superscript II polymerase (Invitrogen). Second strand cDNA, end-repair, A-base addition, and ligation of the Illumina PCR adaptators were performed according to Illumina's protocol. Libraries were then selected for 100 bp cDNA fragments on a 3.5% agarose gel and PCR-amplified using Phusion DNA polymerase (Finnzymes) for 15–18 cycles. PCR products were then purified on a 2% agarose gel and gel-extracted. Each library was quality controlled (product size and concentration) by an Agilent 2100 bioanalyzer. Single-end libraries were sequenced as 100-mers on a Genome Analyzer II flowcell according to Illumina's protocol at a depth of ∼16.2–18.0 million reads per library.
5
Quantitative Gene Expression Analysis by RT-qPCR
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Cells were harvested and lysed in RLT buffer (QIAGEN). RNA extraction was carried out using the RNAeasy micro kit (QIAGEN) according to manufacturer's instructions. Total RNA was retro‐transcribed using the superscript II polymerase (Invitrogen), in combination with random hexamers, oligo dT, and dNTPs (Promega). Transcripts were quantified by real‐time PCR on a 480 LightCycler instrument (Roche). Reactions were carried out in 10 μl, using a master mix (Eurogentec), with the following Taqman Assays primers (Merk), for human samples: B2M (Hs99999907_m1), RPL34 (Hs00241560_m1), HPRT1 (Hs02800695_m1), MX1 (Hs00895608_m1), IFIT3 (Hs00155468_m1), CXCL10 (Hs00895608_m1). The second derivative method was used to determine each Cp and the expression of genes of interest relative to the housekeeping genes (B2M, HPRT, RPL34) was quantified.
6
Transcriptome Analysis Using Affymetrix Microarrays
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Cells were lysed in TRIZOL (Invitrogen, Frederick, MD, USA) and total RNA was further purified with the QIAGEN RNeasy kit following manufacturer’s instructions. RNA was quantified on the NanoDrop ND-1000 and quality checked with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Five µg of total RNA were retrotranscribed from a T7-oligo(dT) primer with the SuperScript II polymerase (Invitrogen). cDNA was then purified on affinity columns and in vitro transcribed with the T7 RNA polymerase and a biotinylated dUTP. Labeled cRNA was purified on affinity columns and quantified on the NanoDrop ND-1000. Twenty µg of cRNA were fragmented and quality checked with the 2100 Bioanalyzer (Agilent Technologies).The biotinylated cRNA was hybridized to the Affimetrix HGU133 Plus 2.0 array, containing about 55,000 probe sets and open reading frames from the H. sapiens genome databases GenBank, dbEST and RefSeq. Chips were washed and scanned on the Affymetrix Complete GeneChip® Instrument System, generating digitized image data (DAT) files.
7
RNA Extraction and RT-PCR Protocol
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Total RNA was extracted from ~50 adult flies with Tri-Reagent (Sigma, St Louis, MO) following the manufacturer’s instructions. Contaminating DNA was degraded by RNase-free DNase I (Thermo Scientific, Waltham, MA). Reverse transcription was performed with Superscript II polymerase (Invitrogen, Carlsbad, CA) following the manufacturer’s guidelines. Finally, Gotaq polymerase (Promega, Fitchburg, WI) was used for PCR amplification with primer sequences and conditions as described in supplementary material Table S2 .
8
Reverse Transcription and qPCR Protocol
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Reverse-transcription was performed on 200ng of RNA using Superscript II polymerase (Invitrogen Cat#18064022) and RNaseOUT ribonuclease inhibitor (Invitrogen Cat#10777019). qPCR was performed on 20ng of cDNAs using Power SYBR Green PCR Master Mix (Applied Biosystems Cat#4367659) and 6pmol of each primer, on a QuantStudio 12K Flex or a ViiA 7 (ThermoFisher Scientific). Primers (Eurofins) sequences are provided in S1 Table . Gene expression was expressed on a Log10 scale of relative expression to the reference Tbp gene.
9
Transcriptome Analysis of PTEC Response
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UA-containing (100 μg/mL) cell culture medium was added to stimulate 2 × 105 preseeded PTECs. After 0, 24, and 48 h of incubation, the supernatants were discarded, and 200 μL of TRIzol reagent (Invitrogen, USA) was added to each of the triplicates for the three groups.
Total RNA was extracted by using TRIzol and a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol with DNase treatment. RNA quality was evaluated with an Agilent 2100QC Bioanalyzer (Agilent Technologies). All RNA samples had RNA integrity numbers (RINs) greater than 8.0. Briefly, 2 μg of total RNA was subjected to ribosomal RNA removal using a Ribo-Zero Magnetic kit (Epicenter, Illumina, USA), 300 ng of rRNA-depleted RNA was fragmented, and cDNA was synthesized using random primers (Takara, Japan) and SuperScript II polymerase (Invitrogen). Illumina TruSeq adaptors were added to the second strand and further amplified with P5/P7 primers. Fragments 350–500 bp in length were recovered with a gel extraction kit. All libraries were quantified and pooled at concentrations of 2 nM for sequencing on a NovaSeq system (150PE; Illumina, USA).
Total RNA was extracted by using TRIzol and a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol with DNase treatment. RNA quality was evaluated with an Agilent 2100QC Bioanalyzer (Agilent Technologies). All RNA samples had RNA integrity numbers (RINs) greater than 8.0. Briefly, 2 μg of total RNA was subjected to ribosomal RNA removal using a Ribo-Zero Magnetic kit (Epicenter, Illumina, USA), 300 ng of rRNA-depleted RNA was fragmented, and cDNA was synthesized using random primers (Takara, Japan) and SuperScript II polymerase (Invitrogen). Illumina TruSeq adaptors were added to the second strand and further amplified with P5/P7 primers. Fragments 350–500 bp in length were recovered with a gel extraction kit. All libraries were quantified and pooled at concentrations of 2 nM for sequencing on a NovaSeq system (150PE; Illumina, USA).
10
High-throughput BCR sequencing from sorted B cells
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Antigen-specific B cells were sorted into OL-1 lysis buffer (Qiagen) and mRNA was extracted using an Oligotex Direct mRNA mini kit (Qiagen). RNA was then concentrated in a centrifugal concentrator (Millipore) and reverse transcription was performed using oligo-dT priming and Superscript II polymerase (Invitrogen). Multiplex 5′ PCR was performed to amplify the IgG HC; reactions proceeded for 35 cycles using an annealing temperature of 48 °C. Primers were derived from ref. 7 (link), and include a five- to nine-nt barcode (all barcodes have a minimum three nt difference), and ‘XLR' sequences for 454 sequencing (Supplementary Table 5 ). PCR products were resolved on a 1.5% agarose gel, and the appropriate bands were excised and extracted. Antigen-specific samples were combined so that each sorted B cell would be sequenced at least 10-fold, given estimates of sequencer read outputs.
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