12/29/2023 0 Comments Dudchenko juicebox youtube![]() In this step, we evaluate the library yield and determine the size distribution of the libraries. Indices are added by PCR and a bead purification and size selection yield Illumina compatible libraries. Intra-aggregate ligation to capture contacts is performed, followed by cross-link reversal and DNA purification.Įnd repair, adapter ligation, and purification steps result in the template for the final stage.Ī streptavidin enrichment step allows capturing the products from the proximity ligation step. Digestion The cross-linked chromatin is digested using two restriction enzymes.Įnd-polishing, ligation of a biotinylated oligonucleotide bridge.In this step, the amount of chromatin obtained, as well as the degree of digestion, is assessed to ensure the success of the library prep. Chromatin is fixed in place using formaldehyde.Ĭhromatin is then released by lysing the cells. ![]() Sample quantification and aliquoting if necessary. The library preparation consists of 5 stages: If the samples pass the reception control, we will inform you and the samples will be queued for library prep. If any of those steps fail, we will contact you. During the protocol, there are a number of additional QC steps. In the case of Hi-C projects, the primary RC consists of the quantification of the available tissue. If the samples fail this quality control step, we will contact you to discuss possible options. Once your samples arrive at NGI, we start by performing a reception control step in which we make sure the sample meets our requirements. If you can not carry out these steps, or your samples do not meet the requirements, please contact us. If possible, keep the mortar on dry ice during the whole procedure. Use liqN2 to pre-cool mortar, spatulas, and tubes. Add more liqN2 if you notice the sample is melting during grinding. The sample should remain frozen through the whole grinding and transfer process. Transfer using a pre-cooled spatula to a pre-cooled tube. Grind the sample using the pestle until it resembles a flour-like powder. Add some liquid nitrogen (liqN2), make sure the sample is completely frozen. The most important aspect in ensuring high sample quality is keeping the tissue frozen, it may not be re-thawed once it has been frozen.įor tissue grinding, the sample should be deposited in a pre-cooled mortar.
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