CHROMATIN

Rating: Not Rated Archive Warning: Graphic Depictions Of Violence Category: F/M, Gen, M/M Fandom: Sherlock (TV) Relationship: Sherlock Holmes/John Watson, Established Relationship(s), Molly Hooper/Greg Lestrade Character: Sherlock Holmes, John Watson, Greg Lestrade, Molly Hooper, Mycroft Holmes, Sally Donovan, Mrs. Hudson, Original Female Character(s), Original Male Character(s) Additional Tags: Murder Mystery, Case Fic, Alpha/Omega, Alpha Sherlock, Omega John, Angst with a Happy Ending, Angst and Fluff and Smut, Canon-Typical Violence, Post-Reichenbach, Established Sherlock Holmes/John Watson, Temporary Character Death, Additional Warnings Apply, Additional Warnings In Author's Note, BAMF John Watson, I have probably gotten some tags wrong., Post Mpreg, Parentlock, Warnings for possible triggers., Feels, Domestic Fluff, Fluff and Angst, Sign Language, Deaf Character Series: Part 2 of Morpholgy Stats: Published: 2015-02-08 Updated: 2018-01-28 Chapters: 24/? Words: 123799

The Bioruptor is the latest innovation in shearing and represents a new breakthrough as all-in-one shearing system that delivers optimal and reproducible chromatin shearing while preserving high protein integrity.
Visit our website to learn more about our different models of Bioruptor and their features.
The shearing device of choice for chromatin preparation for ChIP 9 Obtain perfect fragment size for high quality ChIP Magnetic beads for more reproducible results 9 Keep the epitopes accessible to the antibody 9 Get consistent results from different samples and runs 9 Benefit from the optimal kit for your specific experimental needs

I.I Chromatin preparation
A successful chromatin preparation relies on the optimization of cross-linking, cell lysis and sonication.Our Chromatin EasyShear Kits together with the Bioruptor ultrasonicator combine efficient cell lysis and chromatin shearing leading to consistent results.
Each Chromatin EasyShear Kit provides optimized reagents and a thoroughly validated protocol according to your specific experimental needs.SDS concentration is adapted to each workflow taking into account target-specific requirements.
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I.II Chromatin immunoprecipitation kits
Diagenode's optimized, high performance ChIP kits are the result of over 15 years of research and development.We provide a flexible platform that addresses specific research needs with a vast array of optimized ChIP kits.In addition, we provide the industry's most validated ChIP and ChIP-seq antibodies using a large variety of validation methods.
Which ChIP kit is right?

I.III Library preparation kits
Diagenode's library preparation kits have been extensively validated with ChIP-seq samples.The MicroPlex Library Preparation Kit, which uses a simple 3-step protocol, is an optimal choice for library preparation, especially for very low inputs of DNA down to 50 pg.Moreover, the kits are available with either single or dual index options.

How the MicroPlex Library Preparation Kit Works
Microplex workflow -protocol with dual indexes.
An input of 50 pg to 50 ng of fragmented dsDNA is converted into sequencing-ready libraries for Illumina ® NGS platforms using a fast and simple 3-step protocol.

II. Tagmentation-based technologies
Next generation sequencing technology in the last decade has allowed higher sample throughput at a significantly reduced cost, necessitating the development of novel sequencing applications and methods.In one such method, tagmentation, a modified, hyperactive transposase (Tn5) cuts double-stranded DNA and simultaneously ligates linker sequences to both ends in short reaction time.
The high efficiency of this approach reduces both the input required as well as hands-on time compared to ligation-based library preparation methods.

ATAC-seq Workflow
The ATAC-seq protocol consists of 3 steps: nuclei preparation, tagmentation and library amplification.First, the cells undergo the lysis, ending with the crude nuclei.Then, the nuclei are incubated with a Tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors.
Finally, the generated libraries are amplified and can be used for sequencing.High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.
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II.II ChIPmentation
Diagenode's unique ChIPmentation technology, based on tagmentation, enables the integration of library preparation during ChIP itself using a transposase loaded with sequencing adaptors.Unlike standard library preparation techniques that require many steps, ChIPmentation incorporates an easier and shorter protocol.Moreover, this reduced number of steps allows successful IPs on lower amounts of chromatin, which makes it ideal to analyze numerous histone marks on each chromatin sample.Optimized solutions for: • Low cell number -µChIPmentation for Histones (C01011011) for as little as 10,000 cells per reaction, optimized protocol for FACS sorted cells • Standard cell number -ChIPmentation Kit for Histones (C01011009) • Library preparation using tagmentation -TAG Kit for ChIPmentation (C01011030) can be used with any ChIP protocol • Multiplexing -Primer indexes for tagmented libraries: 24 UDI for tagmented libraries, Set I -III (C0101134, C01011036, C01011037) Separately available products: • Standalone Tn5 transposase: Tagmentase loaded (C01070012) and Tagmentase unloaded (C01070010) • Tagmentation Buffer (1x) (C01019042) • ChIP-seq grade antibodies

ChIPmentation Workflow
ChIPmentation: after cell collection, the DNA and associated proteins are crosslinked in living cells using formaldehyde.Then cells are lysed and chromatin is sheared by sonication (Bioruptor) to obtain the fragments between 100 -600 bp.Crosslinked protein-chromatin complexes of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody.
Tagmentase loaded with sequencing adaptors is added enabling one-step library preparation: the tagmentase introduces sequencing-compatible adaptors in a single-step reaction directly on beadbound chromatin.
After stripping, end-repair and reverse cross-linking, the generated libraries can be amplified and used for sequencing.

II.III CUT&Tag
CUT&Tag-sequencing (Cleavage Under Targets and Tagmentation) is a new chromatin profiling method providing high quality sequencing data from low starting amount.This alternative to ChIP-seq method, combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.Diagenode's iDeal CUT&Tag kit for Histones provides an optimized protocol for a rapid chromatin profiling on histone marks.The protocol is optimized for native cells (10,000-300,000 cells per reaction) and can be completed within 2.5 days.For a complete CUT&Tag protocol the following items must be purchased: • iDeal CUT&Tag for Histones C01070020 -including all reagents for CUT&Tag workflow (buffers, pA-Tn5, CoA beads, DNA purification) • Antibody package for CUT&Tag (anti-rabbit C01070022 or antimouse C01070023) -including the secondary antibody, positive and negative control antibodies and primers • Antibody recognizing the protein of interest -choose one of Diagenode's validated antibodies • Primer indexes for tagmented libraries -for multiplexing up to 72 samples Separately available products: • Fusion protein -compatible with CUT&Tag, but also other applications like ACT-seq, CoBATCH, TIP-seq and more.
(Assay for Transposase-Accessible Chromatin using sequencing) allows for assessing genome-wide chromatin accessibility.The technology is based on the use of the transposase Tn5 which cuts exposed open chromatin and simultaneously incorporates adapters for subsequent amplification and sequencing.• Assess open regions of chromatin at single nucleotide resolution • Gain insight into gene regulation and understand open chromatin signatures • Determine nucleosome positions at single nucleotide resolution