Variable allelic expression of imprinted genes at the Peg13, Trappc9, Ago2 cluster in single neural cells

Fig 1 5' RACE - Sequence tracks and analysis of alternative Trappc9 transcriptional start sites

Fig 2 and suppl fig 3- Brain and Kidney tissue or Neural stem cells isolated from the Hippocampus region of newborn mice generated from a C57BL/6 (female) and a Cast/EiJ (male) cross (and its reciprocal cross) was used to determine allelic bias expression. A SNP located within an exon of Peg13, Trappc9, Ago2, Chrac1 and Kcnk9 was identified and amplified via pyrosequencing PCR with the percentage of SNP identification used to determine allele expression percentages. Additionally, Pyrorun sequences of reverse transcribed RNA from Trappc9 expression in different tissues. A hybrid cross between C57BL/6 and JF1 mouse was used to generate hybrid pups that were used to determine allele specificity of Trappc9 expression in Kidney and brain tissues.

Figs 3, 4 & 5- Single neural stem cells were isolated from the Hippocampus of newborn mice generated from a hybrid cross. Some of these cells were differentiated In vitro and either the NSC or differentiated neurons were lysed and underwent a reverse transcription. The newly formed cDNA was used as a template to amplify expressed Peg13, Trappc9 or Ago2 transcripts which was then sent for Sanger sequencing. A SNP located within the exon was used to determine whether the transcript from that cell was generated from the maternal or paternal allele.

Fig 6- Brain-specific regulatory elements were cloned into a pGL [Luc] vector containing a Trappc9 promoter. These newly generated plasmids were then transfected into either primary neuron or fibroblast cultures alongside a Renilla vector for normalization using Lipofectamine as a transfection reagent. After 48 hours the cells were lysed and analyzed using a Glomax illuminator to determine their impact on Luciferase expression compared to that of the pGL vector containing just the Trappc9 promoter. Additionally, Plasmid vectors that were used for transfection of primary neurons to determine the impact of brain-specific regulatory elements on transcription. Plasmids can be visualized using the free software pdraw.32 downloaded from http://acaclone.com/download/install.htm

Suppl fig 2- Single Neural stem cells and in vitro differentiated neurons isolated from Mouse Hippocampus tissue underwent reverse transcription and a qPCR reaction intended to amplify cDNA of genes associated with specific neural cell types as a method of detecting cell fate and whether this had an impact on allele-specific expression and differential methylation.

Suppl fig 4 & 5- DNA isolated from Neural stem cells was bisulfite converted for downstream identification of methyl group presence. CpG islands located at or near the promoters of the Peg13, Trappc9, Ago2, Chrac1 and Kcnk9 genes were amplified and cloned into a TOPO vector. The cloned segments were Sanger sequenced and compared to the original non-bisulfite converted sequence using the Quantification for methylation analysis (QUMA) tool to determine which CG dinucleotides were methylated and which weren't. Pyroruns determining methylation frequency at the CpG islands of these genes can be found in a separate upload on Zenodo.