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Rta version 2

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RTA version 2.4.11 is a software component that enables real-time analysis of data generated during DNA sequencing runs on Illumina sequencing instruments. It is responsible for processing raw sequencing data and producing base call files for downstream analysis.

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Lab products found in correlation

Hiseq 3000 4000 sequencing kit, by Illumina (9 mentions) Hiseq 4000, by Illumina (9 mentions) Hiseq 3000 4000 pe cluster kit, by Illumina (7 mentions) Cbot and hiseq 3000 4000 pe cluster kit, by Illumina (4 mentions) Mirneasy mini kit, by Qiagen (4 mentions) Nextseq 500 high output kit 75 cycles, by Illumina (4 mentions) Sureselect human all exon v5 utrs 75 mb kit, by Agilent Technologies (3 mentions) Sureselect post capture indexing forward and index pcr reverse primers, by Agilent Technologies (3 mentions) Qubit fluorometry, by Thermo Fisher Scientific (3 mentions) Truseq rna sample prep kit v2, by Illumina (3 mentions) Fragment analyzer, by Agilent Technologies (3 mentions) Standard sensitivity ngs fragment analysis kit, by Agilent Technologies (3 mentions) Bioanalyzer dna 1000 chip, by Thermo Fisher Scientific (2 mentions) Hiseq 2000, by Illumina (2 mentions) Paxgene blood rna tube, by Qiagen (2 mentions) Truseq rna access library prep kit, by Illumina (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Truseq stranded mrna library prep kit high throughput, by Illumina (2 mentions) Nextseq 500, by Illumina (2 mentions) Rna 6000 nano chip, by Agilent Technologies (2 mentions)

24 protocols using rta version 2

1

Single-cell RNA-seq Analysis Pipeline

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Single-cell suspensions were counted using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter, Brea, CA, USA) and ~5000 cells were loaded into the 10X Genomics Chromium system to generate Gel Beads-In-Emulsion (GEMs). Reverse transcription was performed using Chromium Single Cell 3’ Reagent Kits (v2, 10X Genomics, Pleasanton, CA, USA). Standard Illumina sequencing primers and a unique i7 Sample index were added to each cDNA for library construction. Libraries were sequenced using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling was performed using Illumina’s RTA version 2.7.3. Cell Ranger (v3.0) (10X Genomics) was used for alignment to the mouse genome version mm10. R (v3.6.0) and Seurat (v3.1.3)35 (link) were used for further analysis following the standard workflow. For quality control and filtering, cells with detected genes fewer than 200 or >8000, or with mitochondrial gene content >50% were excluded. For Ximerakis’s scRNA-seq dataset, R (v4.0.3) and Seurat (v4.0.2) were used. Gene set enrichment analysis was performed using software GSEA (v4.0.3)53 (link). The interactive website for scRNA-seq data was generated using ShinyCell (v2.1.0)54 .
Zhang X., Pearsall V.M., Carver C.M., Atkinson E.J., Clarkson B.D., Grund E.M., Baez-Faria M., Pavelko K.D., Kachergus J.M., White T.A., Johnson R.K., Malo C.S., Gonzalez-Suarez A.M., Ayasoufi K., Johnson K.O., Tritz Z.P., Fain C.E., Khadka R.H., Ogrodnik M., Jurk D., Zhu Y., Tchkonia T., Revzin A., Kirkland J.L., Johnson A.J., Howe C.L., Thompson E.A., LeBrasseur N.K, & Schafer M.J. (2022). Rejuvenation of the aged brain immune cell landscape in mice through p16-positive senescent cell clearance. Nature Communications, 13, 5671.
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2

Whole-Exome Sequencing of Genetic Disorders

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Whole-exome sequencing (WES) was performed on the proband and her parents at a CLIA-certified laboratory (Mayo Clinic) following a standard procedure summarized as follows: Paired-end libraries were prepared following Agilent’s (Santa Clara, CA, USA) protocol. Whole-exon capture was carried out following the protocol for Agilent’s SureSelect Human All Exon v5 + UTRs 75 MB kit. The purified capture products were amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. The concentration and size distribution of the completed captured libraries were determined on Qubit (Invitrogen, Carlsbad, CA, USA) and Agilent Bioa-nalyzer DNA 1000 chip. Sequencing was performed following Illumina’s standard protocol in the Illumina (San Diego, CA, USA) cBot and HiSeq 3000/4000 PE Cluster Kit (average coverage ~80×). The flow cells were sequenced as 150 × 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling was performed using Illumina’s RTA version 2.7.3.
Kotwal A., Ferrer A., Kumar R., Singh R.J., Murthy V., Schultz-Rogers L., Zimmermann M., Lanpher B., Zimmerman K., Stabach P.R., Klee E., Braddock D.T, & Wermers R.A. (2020). Clinical and Biochemical Phenotypes in a Family With ENPP1 Mutations. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 35(4), 662-670.
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3

Whole Exome Sequencing Workflow

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DNA-sequencing was performed by Mayo Clinic Core facility using Sure Select XT Whole Exon Capture v5 + UTRs 75 MB and Illumina HiSeq 4000 Paired-End Sequencing. Paired-end libraries were prepared using 1.0 μg of genomic DNA following the manufacturer’s protocol (Agilent) using the Agilent Bravo liquid handler. Whole exon capture was carried out using 750 ng of the prepped library following the protocol for Agilent’s SureSelect Human All Exon v5 + UTRs 75 MB kit. The purified capture products are then amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. The concentration and size distribution of the completed libraries was determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Libraries were sequenced at an average coverage of ~250× following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 150 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kits and HCS v3.3.52 collection software. Base-calling is performed using Illumina’s RTA version 2.7.3.
Zhuang Y., Grainger J.M., Vedell P.T., Yu J., Moyer A.M., Gao H., Fan X.Y., Qin S., Liu D., Kalari K.R., Goetz M.P., Boughey J.C., Weinshilboum R.M, & Wang L. (2021). Establishment and characterization of immortalized human breast cancer cell lines from breast cancer patient-derived xenografts (PDX). NPJ Breast Cancer, 7, 79.
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4

Illumina Paired-End Sequencing Protocol

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Libraries were sequenced at 60,000 fragment reads per cell following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling was performed using Illumina’s RTA version 2.7.3.
Chikkamenahalli L.L., Jessen E., Bernard C.E., Ip W.K., Breen-Lyles M., Cipriani G., Pullapantula S.R., Li Y., AlAsfoor S., Wilson L., Koch K.L., Kuo B., Shulman R.J., Chumpitazi B.P., McKenzie T.J., Kellogg T.A., Tonascia J., Hamilton F.A., Sarosiek I., McCallum R., Parkman H.P., Pasricha P.J., Abell T.L., Farrugia G., Dasari S, & Grover M. (2024). Single cell atlas of human gastric muscle immune cells and macrophage-driven changes in idiopathic gastroparesis. iScience, 27(3), 108991.
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5

Trio Whole Exome Sequencing Analysis

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WES from the second miscarriage and both parents was performed at the Clinical Genomics Laboratory (Mayo Clinic). Paired-end libraries were prepared using 1.0 µg of genomic DNA using the Agilent Bravo liquid handler (Agilent) as indicated by the manufacturer. Whole exon capture was carried out using 750 ng of the prepped library following the protocol for Agilent's SureSelect Human All Exon v5 + UTRs 75 MB kit. The purified capture products were amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. Libraries were sequenced at an average coverage of ~80X following Illumina's standard protocol in an Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 150 X 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling is performed using Illumina's RTA version 2.7.3. Data was processed through an in-house bioinformatics pipeline and analyzed using Ingenuity (Qiagen) by the Center for Individualized Medicine (Mayo Clinic).
Ferrer A., Starosta R.T., Ranatunga W., Ungar D., Kozicz T., Klee E., Rust L.M., Wick M, & Morava E. (2020). Fetal glycosylation defect due to ALG3 and COG5 variants detected via amniocentesis: Complex glycosylation defect with embryonic lethal phenotype. Molecular genetics and metabolism, 131(4).
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6

RNA-Seq Analysis of Mouse Liver

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Ground livers from d0 and d21 male (n = 5) mice were used for total RNA isolation by using a miRNeasy Mini Kit (Qiagen) following the manufacturer’s instructions, including an on-column DNase I treatment. RNA was quantified spectrophotometrically. The integrity of RNA was assessed with an Agilent 2100 bioanalyzer in conjunction with the RNA 6000 Nano Kit (Agilent), and a RNA integrity number greater than or equal to nine was used. RNA-Seq was performed at the GSPMC, Medical College of Wisconsin. RNA libraries were prepared by using 200 ng of total RNA according to the manufacturer’s instructions for the Illumina TruSeq RNA v2 kit (Illumina, San Diego, CA, USA), and sequencing was completed on the Illumina HiSeq-2000 with 101 bp paired-end reads. Base calling is performed using Illumina’s RTA version 2.5.2.
Fu Q., Cheung W.A., Majnik A.V., Ke X., Pastinen T, & Lane R.H. (2023). Adverse Maternal Environments Perturb Hepatic DNA Methylome and Transcriptome Prior to the Adult-Onset Non-Alcoholic Fatty Liver Disease in Mouse Offspring. Nutrients, 15(9), 2167.
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7

Bulk RNA-seq Library Preparation and Sequencing

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Generation of raw bulk RNAseq data in the study 2 cohort was previously described6 ,46 . Briefly, libraries were prepared from total RNA using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) and sequenced on Illumina HiSeq 2000 instruments. For study 1, cDNA libraries were prepared using 200 ng of total RNA according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA). The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Libraries were sequenced at six samples per lane, following Illumina’s standard protocol using the HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 ×2 paired-end reads on an Illumina HiSeq 4000 using the HiSeq 3000/4000 sequencing kit and HCS v3.3.20 collection software. Base-calling was performed using Illumina’s RTA version 2.5.2.
Min Y., Wang X., İş Ö., Patel T.A., Gao J., Reddy J.S., Quicksall Z.S., Nguyen T., Lin S., Tutor-New F.Q., Chalk J.L., Mitchell A.O., Crook J.E., Nelson P.T., Van Eldik L.J., Golde T.E., Carrasquillo M.M., Dickson D.W., Zhang K., Allen M, & Ertekin-Taner N. (2023). Cross species systems biology discovers glial DDR2, STOM, and KANK2 as therapeutic targets in progressive supranuclear palsy. Nature Communications, 14, 6801.
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8

Bulk mRNA-seq Library Preparation and Sequencing

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mRNA-seq libraries were prepared using 200 ng of total RNA with a TruSeq RNA Sample Prep Kit v2 (Illumina) as described previously [19 (link)]. Libraries were sequenced at approximately 75 million reads/sample following Illumina's protocol using the Illumina cBot and HiSeq 3000/4000 PE cluster kit. The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 using Hiseq 3000/4000 sequencing kits and HCS v3.3.20 data collection software. Base-calling was performed using Illumina's RTA version 2.5.2.
Ryan Z.C., Craig T.A., Wang X., Delmotte P., Salisbury J.L., Lanza I.R., Sieck G.C, & Kumar R. (2018). 1α,25-dihydroxyvitamin D3 mitigates cancer cell mediated mitochondrial dysfunction in human skeletal muscle cells. Biochemical and biophysical research communications, 496(2), 746-752.
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9

RNA-seq Analysis of P27 Variant

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RNA sequencing was performed from blood RNA from P27 bearing the p.(Trp1787*) variant by first isolating RNA using the miRNeasy Mini Kit (Qiagen) following the standard protocol from blood drawn in a PAXgene Blood RNA Tube (Qiagen). RNA libraries were prepared, and coding regions of the transcriptome were captured by pooling four of the cDNA libraries at 200 ng each according to the manufacturer’s instructions for the TruSeq® RNA Access Library Prep Kit (Illumina)68 (link). Libraries were sequenced at ~65 million fragment reads per sample (4 samples/lane) following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.20 collection software. Base-calling was performed using Illumina’s RTA version 2.5.2. RNA-sequencing analysis was performed using MAP-RSeq69 (link). Reads were aligned to the human genome (hg19) and transcriptome using Tophat270 (link) running Bowtie (v1)71 (link). Gene and exon level read counts were generated using HiSeq72 (link) and BedTools73 (link), respectively. Alignments were visualized using Integrative Genomics Viewer (IGV) (http://software.broadinstitute.org/software/igv/).
Cousin M.A., Creighton B.A., Breau K.A., Spillmann R.C., Torti E., Dontu S., Tripathi S., Ajit D., Edwards R.J., Afriyie S., Bay J.C., Harper K.M., Beltran A.A., Munoz L.J., Rodriguez L.F., Stankewich M.C., Person R.E., Si Y., Normand E.A., Blevins A., May A.S., Bier L., Aggarwal V., Mancini G.M., van Slegtenhorst M.A., Cremer K., Becker J., Engels H., Aretz S., MacKenzie J.J., Brilstra E., van Gassen K.L., van Jaarsveld R.H., Oegema R., Parsons G.M., Mark P., Helbig I., McKeown S.E., Stratton R., Cogne B., Isidor B., Cacheiro P., Smedley D., Firth H.V., Bierhals T., Kloth K., Weiss D., Fairley C., Shieh J.T., Kritzer A., Jayakar P., Kurtz-Nelson E., Bernier R.A., Wang T., Eichler E.E., van de Laar I.M., McConkie-Rosell A., McDonald M.T., Kemppainen J., Lanpher B.C., Schultz-Rogers L.E., Gunderson L.B., Pichurin P.N., Yoon G., Zech M., Jech R., Winkelmann J., Beltran A.S., Zimmermann M.T., Temple B., Moy S.S., Klee E.W., Tan Q.K, & Lorenzo D.N. (2021). Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome. Nature genetics, 53(7), 1006-1021.
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10

RNA-seq Analysis of P27 Variant

Cited 2 times
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RNA sequencing was performed from blood RNA from P27 bearing the p.(Trp1787*) variant by first isolating RNA using the miRNeasy Mini Kit (Qiagen) following the standard protocol from blood drawn in a PAXgene Blood RNA Tube (Qiagen). RNA libraries were prepared, and coding regions of the transcriptome were captured by pooling four of the cDNA libraries at 200 ng each according to the manufacturer’s instructions for the TruSeq® RNA Access Library Prep Kit (Illumina)68 (link). Libraries were sequenced at ~65 million fragment reads per sample (4 samples/lane) following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.20 collection software. Base-calling was performed using Illumina’s RTA version 2.5.2. RNA-sequencing analysis was performed using MAP-RSeq69 (link). Reads were aligned to the human genome (hg19) and transcriptome using Tophat270 (link) running Bowtie (v1)71 (link). Gene and exon level read counts were generated using HiSeq72 (link) and BedTools73 (link), respectively. Alignments were visualized using Integrative Genomics Viewer (IGV) (http://software.broadinstitute.org/software/igv/).
Cousin M.A., Creighton B.A., Breau K.A., Spillmann R.C., Torti E., Dontu S., Tripathi S., Ajit D., Edwards R.J., Afriyie S., Bay J.C., Harper K.M., Beltran A.A., Munoz L.J., Rodriguez L.F., Stankewich M.C., Person R.E., Si Y., Normand E.A., Blevins A., May A.S., Bier L., Aggarwal V., Mancini G.M., van Slegtenhorst M.A., Cremer K., Becker J., Engels H., Aretz S., MacKenzie J.J., Brilstra E., van Gassen K.L., van Jaarsveld R.H., Oegema R., Parsons G.M., Mark P., Helbig I., McKeown S.E., Stratton R., Cogne B., Isidor B., Cacheiro P., Smedley D., Firth H.V., Bierhals T., Kloth K., Weiss D., Fairley C., Shieh J.T., Kritzer A., Jayakar P., Kurtz-Nelson E., Bernier R.A., Wang T., Eichler E.E., van de Laar I.M., McConkie-Rosell A., McDonald M.T., Kemppainen J., Lanpher B.C., Schultz-Rogers L.E., Gunderson L.B., Pichurin P.N., Yoon G., Zech M., Jech R., Winkelmann J., Beltran A.S., Zimmermann M.T., Temple B., Moy S.S., Klee E.W., Tan Q.K, & Lorenzo D.N. (2021). Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome. Nature genetics, 53(7), 1006-1021.
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