Maxima rt polymerase

Manufactured by Thermo Fisher Scientific

Maxima RT polymerase is a thermostable reverse transcriptase enzyme that catalyzes the synthesis of complementary DNA (cDNA) from an RNA template. It is designed for high-performance reverse transcription and is suitable for a wide range of applications, including gene expression analysis, cDNA library construction, and RT-PCR.

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Lab products found in correlation

Nextseq 500, by Illumina (6 mentions) Ultra 2 fs kit, by New England Biolabs (3 mentions) Nextera xt kit, by Illumina (2 mentions) Neb ultra 2 fs kit, by New England Biolabs (1 mentions)

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Maxima RT

8 protocols using maxima rt polymerase

Poly(A)-RNA Bulk Sequencing Protocol

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Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously^{36 (link)}. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adapter. Ends of the cDNAs were extended by a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adapter. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing, a TruSeq adapter was ligated and 3'-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al.^{36 (link)}, the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2. Data was processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables. Reference genome (GRCm38) was used for alignment^{37 (link)}. Transcript and gene definitions were used according to the GENCODE Version M25. Heatmaps shown display the log2 fold change.

Randriamanantsoa S., Papargyriou A., Maurer H.C., Peschke K., Schuster M., Zecchin G., Steiger K., Öllinger R., Saur D., Scheel C., Rad R., Hannezo E., Reichert M, & Bausch A.R. (2022). Spatiotemporal dynamics of self-organized branching in pancreas-derived organoids. Nature Communications, 13, 5219.

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Bulk RNA-seq library preparation

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Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously.¹¹⁵ (link) Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. Ends of the cDNAs were extended by a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adaptor. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing a TruSeq adapter was ligated and 3’-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al.,¹¹⁵ (link) the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.

Fischer A., Lersch R., de Andrade Krätzig N., Strong A., Friedrich M.J., Weber J., Engleitner T., Öllinger R., Yen H.Y., Kohlhofer U., Gonzalez-Menendez I., Sailer D., Kogan L., Lahnalampi M., Laukkanen S., Kaltenbacher T., Klement C., Rezaei M., Ammon T., Montero J.J., Schneider G., Mayerle J., Heikenwälder M., Schmidt-Supprian M., Quintanilla-Martinez L., Steiger K., Liu P., Cadiñanos J., Vassiliou G.S., Saur D., Lohi O., Heinäniemi M., Conte N., Bradley A., Rad L, & Rad R. (2023). In vivo interrogation of regulatory genomes reveals extensive quasi-insufficiency in cancer evolution. Cell Genomics, 3(3), 100276.

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Bulk RNA-seq Library Preparation with UMIs

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Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously^{78 (link)}. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adapter. 5’-Ends of the cDNAs were extended by a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adapter. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing a TruSeq adapter was ligated and 3’-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al.^{78 (link)}, the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 59 cycles for the cDNA in read1 and 18 cycles for the barcodes and UMIs in read2. Data was processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables^{79 (link)}. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the GENCODE version M25. Differential expression analysis was performed using R and DESeq2 (1.38.0). A P value of <0.05 was used to determine differentially expressed genes. The data

Linnerbauer M., Beyer T., Nirschl L., Farrenkopf D., Lößlein L., Vandrey O., Peter A., Tsaktanis T., Kebir H., Laplaud D., Oellinger R., Engleitner T., Alvarez J.I., Rad R., Korn T., Hemmer B., Quintana F.J, & Rothhammer V. (2023). PD-L1 positive astrocytes attenuate inflammatory functions of PD-1 positive microglia in models of autoimmune neuroinflammation. Nature Communications, 14, 5555.

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Bulk 3' RNA-seq Library Prep

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Library preparation for bulk 3′-sequencing of poly(A)-RNA was done as described previously^{67 (link)}. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adapter. 5′ ends of the cDNAs were extended by a template switch oligo (TSO) and after pooling of all samples full-length cDNA was amplified with primers binding to the TSO-site and the adapter. cDNA was tagmented with the Nextera XT kit (Illumina) and 3′-end-fragments finally amplified using primers with Illumina P5 and P7 overhangs. The library was sequenced on a NextSeq. 500 (Illumina) with 16 cycles for the barcodes and UMIs in read1 and 65 cycles for the cDNA in read2.

Hidalgo-Sastre A., Lubeseder-Martellato C., Engleitner T., Steiger K., Zhong S., Desztics J., Öllinger R., Rad R., Schmid R.M., Hermeking H., Siveke J.T, & von Figura G. (2020). Mir34a constrains pancreatic carcinogenesis. Scientific Reports, 10, 9654.

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Bulk RNA-seq Library Preparation and Analysis

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The initial library preparation for bulk‐sequencing of poly(A)‐RNA was performed as described previously.^[⁹⁵^] Briefly, the barcoded cDNA of each sample was generated using Maxima RT polymerase (Thermo Fisher Scientific) and oligo‐dT primer containing barcodes, unique molecular identifiers (UMIs) as well as an adaptor. cDNA ends were extended using a template switch oligo (TSO), following full‐length cDNA amplification using primers that bind to the TSO‐site and the adaptor. Further cDNA fragmentation was performed using NEB UltraII FS kit. After end‐repair and A‐tailing a TruSeq adapter was ligated and 3’‐end‐fragments were finally amplified with Illumina using the P5 and P7 overhang primers. In comparison to Parekh et al. (2016),^[⁹⁵^] the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 67 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2. Data was processed using the published Drop‐seq pipeline (v1.0) to generate sample‐ and gene‐wise UMI tables.^[⁹⁶^] Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the GENCODE Version M25.

Arnold F., Mahaddalkar P.U., Kraus J.M., Zhong X., Bergmann W., Srinivasan D., Gout J., Roger E., Beutel A.K., Zizer E., Tharehalli U., Daiss N., Russell R., Perkhofer L., Oellinger R., Lin Q., Azoitei N., Weiss F., Lerch M.M., Liebau S., Katz S., Lechel A., Rad R., Seufferlein T., Kestler H.A., Ott M., Sharma A.D., Hermann P.C, & Kleger A. (2021). Functional Genomic Screening During Somatic Cell Reprogramming Identifies DKK3 as a Roadblock of Organ Regeneration. Advanced Science, 8(14), 2100626.

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Bulk-sequencing of poly(A)-RNA

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Library preparation for bulk‐sequencing of poly(A)‐RNA was done as described previously.^[³⁵^] Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo‐dT primer containing barcodes, unique molecular identifiers (UMIs), and an adaptor. 5′‐Ends of the cDNAs were extended by a template switch oligo (TSO) and full‐length cDNA was amplified with primers binding to the TSO‐site and the adaptor. NEB UltraII FS kit (New England Biolabs) was used to fragment cDNA. After end repair and A‐tailing a TruSeq adapter was ligated and 3’‐end‐fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al. (2016), the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2. Data were processed using the published Drop‐seq pipeline (v1.0) to generate sample‐ and genewise UMI tables. Principal component analysis and heat map generation was carried out by the R package Pheatmap.

Bozoglu T., Lee S., Ziegler T., Jurisch V., Maas S., Baehr A., Hinkel R., Hoenig A., Hariharan A., Kim C.I., Decker S., Sami H., Koppara T., Oellinger R., Müller O.J., Frank D., Megens R., Nelson P., Weber C., Schnieke A., Sperandio M., Santamaria G., Rad R., Moretti A., Laugwitz K., Soehnlein O., Ogris M, & Kupatt C. (2022). Endothelial Retargeting of AAV9 In Vivo. Advanced Science, 9(7), 2103867.

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Single-cell RNA-sequencing of Organoids

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Approximately five confluent organoid wells were used to harvest RNA for RNA‐sequencing (RNAseq). Organoids (ID188 passage 7, ID211 passage 6) were processed as abovementioned, and the cell pellet was resuspended in RLT buffer supplemented with β‐mercaptoethanol (Parekh et al, 2016 (link)). Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher Scientific) using oligo‐dT primer containing barcodes, unique molecular identifiers (UMIs), and an adaptor. Ends of the cDNAs were extended by a template switch oligo (TSO), and full‐length cDNA was amplified with primers binding to the TSO‐site and the adaptor. The NEB UltraII FS kit was used to fragment cDNA. After end repair and A‐tailing a TruSeq (Parekh et al, 2016 (link)), the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve better cluster recognition. The library was sequenced on a NextSeq (Macosko et al, 2015 (link)). Reference genome (GRCh38) was used for alignment. Transcript and gene definitions were used according to the GENCODE Version M25.

Peschke K., Jakubowsky H., Schäfer A., Maurer C., Lange S., Orben F., Bernad R., Harder F.N., Eiber M., Öllinger R., Steiger K., Schlitter M., Weichert W., Mayr U., Phillip V., Schlag C., Schmid R.M., Braren R.F., Kong B., Demir I.E., Friess H., Rad R., Saur D., Schneider G, & Reichert M. (2022). Identification of treatment‐induced vulnerabilities in pancreatic cancer patients using functional model systems. EMBO Molecular Medicine, 14(4), e14876.

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Bulk 3' poly(A)-RNA sequencing protocol

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Library preparation for bulk 3′-sequencing of poly(A)-RNA was done as described previously^{57 (link)}. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adapter. 5′ ends of the cDNAs were extended by a template switch oligo (TSO) and after pooling of all samples full-length cDNA was amplified with primers binding to the TSO-site and the adapter. cDNA was tagmented with the Nextera XT kit (Illumina) and 3′-end-fragments finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al.^{57 (link)}, the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 65 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.
Data were processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the ENSEMBL annotation release 75.

Lechner M., Engleitner T., Babushku T., Schmidt-Supprian M., Rad R., Strobl L.J, & Zimber-Strobl U. (2021). Notch2-mediated plasticity between marginal zone and follicular B cells. Nature Communications, 12, 1111.

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