6.1 PRESENTACIÓN DE RESULTADOS

2.10.1.1 ChIP DNA Whole Genome Amplification (WGA)

Primary myocyte ChIP chromatin from 2.7.1, including both the KCNMA1 and IgG fractions were amplified using the Sigma Whole Genome Amplification Kit (WGA) as detailed in the manufacturer’s instructions with slight modifications. One microlitre of ChIP DNA was diluted with 9µl of ultrapure water. Two microlitres of library preparation buffer together with 1µl of library stabilisation solution was added to this and heated at 95ºC for two minutes before cooling on ice. One microlitre of library preparation enzyme was then added and the reaction incubated in the thermal cycler for the following:

20 minutes at 16ºC (pre-cooled to this temperature) 20 minutes at 20ºC

20 minutes at 37ºC 5 minutes at 75ºC 4ºC hold

During the incubation a master mix containing 7.5µl of 10x amplification master mix, 47.5µl ultrapure water and 5µl (2 units/µl) of Whole Genome Amplification (WGA) DNA polymerase was prepared. This was added to each sample and incubated in the thermal cycler for the following:

3 minutes at 95ºC Then 20 cycles of: 15 seconds at 95ºC 5 minutes at 65ºC

115 4ºC hold

Ten percent of the amplified DNA was run on an agarose gel to ensure a DNA smear of the correct size was obtained. The remainder of the DNA was purified using Sigma Genelute™ PCR Clean-Up Kit following the manufacturers guidelines, as detailed in section 2.7.5.

2.10.1.2 Re-Amplification of Amplified DNA

One round of amplification did not produce enough DNA for ChIP sequencing experiments, therefore, the amplified DNA was re-amplified using the WGA re- amplification kit to generate the 7.5µg required. The method was as follows: 1µl of amplified DNA was added to 9µl of ultrapure water. As master mix containing 47.5µl ultrapure water, 7.5µl amplification master mix, 2.5units WGA polymerase and dNTPS (10 mM dGTP, 10 mM dCTP, 10 mM dTTP and 10 mM dATP) was prepared. This was added to each sample and incubated in the thermal cycler for the following:

3 minutes at 95ºC Then 20 cycles of: 15 seconds at 95ºC 5 minutes at 65ºC 4ºC hold

Ten percent of the amplified DNA was run on an agarose gel to ensure a DNA smear of the correct size was obtained. The remainder of the DNA was purified using Sigma Genelute™ PCR Clean-Up Kit following the manufacturer’s guidelines, as detailed in section 2.7.5. The DNA was quantified using the nanophotometer by measuring UV absorbtion at 260nm. Using the Beer Lambert Law where an absorbtion of one equates to a concentration of 50μg/ml DNA. The ratio of UV absorbtion at A260 and A280nm was used to assess the purity of the sample. The A260/280 for pure DNA is ~1.8-2.0.

2.10.1.3 ChIP Sequencing End Repair

The ChIP sequencing (ChIP-seq) was carried out by Dr Paul Heath in Sheffield Institute for Translational Neuroscience (SITrAN). End Repair was carried out using the Kapa

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Biosystems Library Preparation Kit according to the manufacturer’s instructions. Briefly, a reaction mix comprising 8μl water, 7μl 10x Kapa end repair buffer, 5μl Kapa end repair enzyme mix and 50μl fragmented DNA was prepared and incubated at 20°C for 30 minutes. Following this 120μl of Agencourt® _AMPure® _{XP Reagent was added to each} reaction and incubated at room temperature for 15 minutes. This reaction was then placed on a magnet until the supernatant was clear. The supernatant was discarded and the beads washed twice with 200μl 80% (v/v) ethanol.

2.10.1.4 Addition of ‘A’ Bases to the 3’ end of the DNA Fragments

This was again carried out using the Kapa Biosystems Library Preparation Kit according to the manufacturer’s instructions. Briefly, a reaction mix comprising the beads (from above), 42μl water, 5μl 10x KAPA A tailing buffer, 3μl KAPA A tailing enzyme was prepared. This was incubated at 30°C for 30 minutes. This reaction was then placed on a magnet until the supernatant was clear. The supernatant was discarded and the beads washed twice with 200μl 80% (v/v) ethanol.

2.10.1.5 Ligate Adapters to DNA Fragments

At this point the protocol was switched to the NEBNext Ultra DNA Library Preparation Kit and the manufacturer’s protocols followed. Briefly, a reaction mix comprising the beads (from above), 15μl Blunt/TA Ligase Master Mix, 2.5μl NEBNext Adaptor for Illumina, 1μl Ligation Enhancer was prepared. This mix was incubated at 20°C for 15 minutes. At this point 3μl USER enzyme was added to the mix and incubated at 37°C for 15 minutes. This reaction was then placed on a magnet until the supernatant was clear. The supernatant was discarded and the beads washed twice with 200μl 80% (v/v) ethanol. The DNA was then eluted from the beads by the addition of 22μl 10mM Tris HCl and incubation at room temperature for two minutes. This reaction was then placed on a magnet until the supernatant was clear. The supernatant was collected.

2.10.1.6 Enrichment of the Adapter-Modified DNA Fragments by PCR.

The following PCR reaction mix was prepared: 20μl DNA (from above), 2.5μl Index Primer/i7 Primer, 2.5μl Universal primer/i5 primer, 25μl NEBNext High Fidelity 2x PCR Master Mix. This was then amplified using the following PCR protocol:

117 30 seconds at 98°C

Then 18 cycles of: 10 seconds at 98°C 30 seconds at 65°C 30 seconds at 72°C Then: 5 minutes at 72°C Hold at 4°C

The DNA was then again purified and eluted in 28μl. The library was then validated using the bioanalyser. The bioanalyser utilises chip based electrophoresis to analyse RNA and DNA, a dye is intercalated into the sample which then runs past a filter the fragments are detected by laser induced fluorescence. Purity, size and concentration of the sample is then calculated by calibration against the ladder which is of known size and concentration. Briefly, 1μl of both the construct and the negative control are loaded onto the bioanalyser and the size purity and concentration of the sample were checked.

2.10.1.7 Cluster Generation by Bridge Amplification

The flow cell surface is coated with single stranded oligonucleotides corresponding to the adaptor sequences which were ligated to the DNA. These single stranded adapter ligated fragments were bound to the surface of the flow cell in the presence of reagents for polymerase based extension. The flow cell and bound DNA then underwent a series of denaturation and extension cycles resulting in localised amplification of single molecules in millions of unique locations across the flow cell surface.

2.10.1.8 Sequencing by Synthesis

The flow cell (from above) which now contains millions of unique clusters was then loaded into the sequencer for automated cycles of extension and imaging.