RNA-seq Analysis of P27 Variant

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-RSeq^{69 (link)}. Reads were aligned to the human genome (hg19) and transcriptome using Tophat2^{70 (link)} running Bowtie (v1)^{71 (link)}. Gene and exon level read counts were generated using HiSeq^{72 (link)} and BedTools^{73 (link)}, respectively. Alignments were visualized using Integrative Genomics Viewer (IGV) (http://software.broadinstitute.org/software/igv/).

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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.

Publication 2021

miRNeasy Mini Kit PAXgene Blood RNA Tube TruSeq® RNA Access Library Prep Kit cBot and HiSeq 3000/4000 PE Cluster Kit HiSeq 4000 HiSeq 3000/4000 sequencing kit RTA version 2.5.2

Blood Cdna libraries Cells Exon Gene Human genome Library Rna sequencing analysis Transcriptome

Corresponding Organization :

Other organizations : Mayo Clinic in Florida, Mayo Clinic, University of North Carolina at Chapel Hill, Duke University Hospital, Duke Medical Center, Medical College of Wisconsin, Yale University, Columbia University, Erasmus MC, University of Bonn, University Hospital Bonn, McMaster University, University Medical Center Utrecht, Spectrum Health, Children's Hospital of Philadelphia, University of Pennsylvania, Driscoll Children's Hospital, Inserm, Nantes Université, Centre National de la Recherche Scientifique, Institut du Thorax, Génétique Médicale & Génomique Fonctionelle, Queen Mary University of London, William Harvey Research Institute, Wellcome Sanger Institute, Cambridge University Hospitals NHS Foundation Trust, Universität Hamburg, University Medical Center Hamburg-Eppendorf, University of California, San Francisco, Boston Children's Hospital, Miami Children's Hospital, University of Washington, Howard Hughes Medical Institute, University of Toronto, Hospital for Sick Children, SickKids Foundation, Technical University of Munich, Helmholtz Zentrum München, General University Hospital in Prague, Charles University, Munich Cluster for Systems Neurology

Top 5 similar protocols

Variable analysis

independent variables

P.(Trp1787*) variant

dependent variables

Gene and exon level read counts
Transcriptome expression

control variables

Blood RNA from P27 individual
RNA isolation using miRNeasy Mini Kit (Qiagen)
RNA libraries preparation and coding regions capture using TruSeq® RNA Access Library Prep Kit (Illumina)
Sequencing at ~65 million fragment reads per sample using Illumina HiSeq 4000
RNA-sequencing analysis using MAP-RSeq

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