Illumina HiSeq 2000 paired end sequencing; GSM2029245: 3PNT.4; Homo sapiens; RNA-Seq

3PNT.4

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Assisted reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable women who carry mtDNA mutations to have a genetically related child with a greatly reduced risk of disease. Here we report for the first time that pronuclear transplantation (PNT) between normally fertilised human zygotes provides an effective approach to preventing transmission of mtDNA disease.  We found that the procedures previously used to perform PNT between abnormally fertilized human zygotes are highly inefficient when applied to those that undergo normal fertilization.  We have therefore developed an alternative approach based on transplanting PN shortly after completion of the second meiotic division rather than shortly before onset of the first mitosis.  This approach promotes highly efficient development to the blastocyst stage without affecting nuclear genome integrity. Furthermore, the expression profile of genes encoded by the nuclear and mitochondrial genomes was indistinguishable from unmanipulated control embryos. Importantly, levels of mtDNA transferred with the nuclear genome are below the threshold for mtDNA disease. Together these data indicate that transplantation of pronuclei early in the first cell cycle holds promise as a safe and effective approach to preventing transmission of mtDNA disease. Overall design: Single-Cell RNA-seq analysis of embryos generated by pronuclear transfer and unmanipulated control embryos The relationship between single cell samples and the embryo from which they were derived is indicated in the sample ''characteristics: sample type'' field.

Towards therapeutic application of pronuclear transfer to prevent transmission of mitochondrial DNA disease

Submitted by Gene Expression Omnibus on 04-JUN-2016

cDNA was generated from single cells using the SMARTer Ultra Low Input RNA kit for Illumina Sequencing–HV (Clontech Laboratories, Inc.) according to maunfacturers’ guidelines. Single cells were picked using 100 micromteer inner diameter Stripper pipette (Origio) and transferred to individual low bind RNAse-free tube containing 0.25 μl RNase inhibitor, 4.75 μl Dilution buffer and 5 microliter nuclease-free water on a -80°C pre-chilled CoolRack (Biocision, CA). Samples were stored at -80°C until ready to be processed. 1 microliter of 3’ SMART CDS Primer II A was added to the sample, mixed well and incubated at 72°C for 3 min. First strand cDNA was synthesised by adding 4microliter 5X First-Strand Buffer, 0.5 microliter 100 mM DTT, 1 microliter 20 mM dNTP mix, 1 microliter  SMARTer IIA Oligonucleotide, 0.5 microliter RNase Inhibitor and 2 microliter SMARTScribe Reverse Transcriptase (100 U/microliter) directly to a tube containing the sample and incubating at 42°C for 2 hours followed by 10 min at 70°C. First strand cDNA was purified by adding 36 microliter of room temperature SPRI Ampure XP beads (Beckman Coulter Genomics), mixing well and incubating at room temperature for 8 min. Tubes were placed on a MagnaBot II Magnetic Separation device (Promega) and allowed to stand until all beads were immobilised into a pellet. The supernatant was removed and discarded. Tubes were briefly spun and any residual liquid was removed. Double stranded cDNA was amplified from the template bound to the beads using Advantage 2 PCR kit (Clontech Laboratories, Inc.). 5 microlite r10X Advantage 2 PCR Buffer, 2 microliter 10 mM dNTP Mix, 2 microliter IS PCR Primer, 2 microliter 50X Advantage 2 Polymerase Mix and 39 microliter Nuclease-Free water were added to the tube containing the sample to give a total volume of 50 microliter. PCR amplification was performed at 95°C for 1 min, followed by 18 cycles of 15 sec at 95°C, 30 sec at 65°C and 6 min at 68°C followed by final extension step of 10 min at 72°C. Amplified cDNA was purified by adding 90 microliter SPRI Ampure XP beads, mixing well and incubating at room temperature for 8 min to allow amplified cDNA bind to the beads. Sample tubes were placed on the magnet and allowed to stand until all beads had been immobilised. Supernatant was removed and discarded and beads were washed twice by adding 200 microliterl freshly prepared 80% ethanol and leaving for 30 sec before discarding the supernatant. Tubes were spun briefly to collect residual liquid. The bead pellet was allowed to air dry. 12 microliter of purification buffer was added to rehydrate the pellet and incubated for 2 min at room temperature. cDNA was eluted by pipetting up and down 10 times before returning the tube to the magnet. The clear supernatant containing the cDNA was removed from the immobilised beads and transferred to a new low-bind tube. cDNA was stored at -80°C until library preparation. cDNA quality was assessed by High Sensitivity DNA assay on an Agilent 2100 Bioanalyser with good quality cDNA showing a broad peak from 300 to 9000 bp. cDNA concentration was measured using QuBit dsDNA HS kit (Life Technologies UK Ltd.) Typical yields from a single cell ranged from 1 ng to 9 ng. In preparation for library generation cDNA was sheared using Covaris S2 to achieve cDNA in 200-500 bp range. 10 μl of cDNA sample and 65 microliter purification buffer was added to Covaris AFA Fiber Pre-Slit Snap Cap microTUBE. cDNA was sheared using the settings 10% Duty, Intensity 5, Burst Cycle 200 for 2 min. Sheared cDNA was transferred to a new 0.2 ml low-bind tube.Libraries were prepared using Low Input Library Prep Kit (Clontech Laboratories, Inc.) according to manufacturer’s instructions. The amount of input cDNA was calculated from the concentration measured by the Bioanalyser assay prior to shearing, taking into account the dilution involved in the shearing step. The appropriate amplification cycle number was selected according to manufacturer’s guidelines. Library quality was assessed by Bioanalyser and the concentration was measured by QuBit assay.  The molar concentration of library was calculated thus: Library molecular weight = average size in bp (from Bioanalyser) x 650 g/mol per bp Molar concentration = library concentration from QuBit/library molecular weight Libraries with a molar concentration greater than 2nM were submitted for 50-bp paired-end sequencing on Illumina HiSeq 2000.

Towards therapeutic application of pronuclear transfer to prevent transmission of mitochondrial DNA disease

Submitter Supplied Information

Samples