Computational protocol: Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)*

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Protocol publication

[…] hPSCs cultivated on MatrigelTM in MEF-CM were harvested, lysed in TRIzol® (Life Technologies) and total RNA was extracted applying NucleoSpin® RNA II Kit (Machery-Nagel). To remove rRNA the Ribo-ZeroTM Kit (Epicenter, Madison, WI) was used. Subsequently, 10 μg of each total RNA sample was mixed with Ambion® ERCC Spike-in Control Mixes (Life Technologies). For sequencing, RNA was prepared according to the SOLiD® Total RNA-Seq Kit (Life Technologies). Briefly, rRNA-free RNA was hydrolyzed into small fragments followed by a phosphorylation step. After adaptor-ligation and hybridization, cDNA was reverse transcribed from the purified RNA-fragments. cDNA fragments of correct length were purified by Agencourt AMPure XP bead purification (Beckman Coulters Genomics, Danvers, MA) and amplified for 12 PCR cycles in a Biometra T3 Thermocycler using barcoded primers. Size distribution and concentration of fragments was determined with an Agilent 2100 Bioanalyzer and its provided chemistry (Agilent Technologies). cDNA fragments were pooled in equimolar ratios and diluted to a concentration of 500 pm (61 pg/μl). In compliance with the E80 scale protocol provided by the manufacturer (Life Technologies), 50 μl of cDNA mixtures were spiked with a fresh oil emulsion, P1 beads, P1 and P2 chemicals in a SOLiD EZ Bead Emulsifier. Amplification of cDNA was conducted in the E80-modus of a SOLiD EZ Bead Amplifier (Life Technologies). Beads, carrying the amplified template DNA, were purified on a SOLiD EZ Bead Enricher (Life Technologies). The purified beads were put upside down onto a SOLiD 6-lane Flowchip for 1 h at 37 °C. The DNA on the Flowchip was sequenced in read length of 75 bases in a 5500xl SOLiD System.Procession and read mapping of the run was carried out with the Lifescope software analysis tool (Life Technologies) and reference genome GRCh37/hg19. Statistical analysis was performed with R (). Normalization and differential expression calculations were carried out using the R packages EDASeq () and DESeq (), respectively. […]

Pipeline specifications

Software tools LifeScope, EDASeq, DESeq
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Genetic Diseases, Inborn
Chemicals Mannose