CVD lecture: Link to UCSC browser session

 

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Task-1: Vista Enhancer Search.

Access Vista Enhancer Browser (https://enhancer.lbl.gov) -> Advanced Search

Q1: Determine the number of elements driving any heart enhancer activity (both mouse and human sequences).

Q2: Determine the number of all human genomic elements that have been tested for heart enhancer activity.

Q3: How many of the elements determined in Q2 show heart activity?

 

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Task-2: Upload of Vista Enhancer Elements to UCSC Browser.

-> upload mm10 Vista enhancer coordinates to UCSC browser session in BED format.

-> access excel file “Vista_Enhancer_Browser_coordinates” (available here), derived (copied) from the Vista Enhancer Browser (format obtained under task1).

-> copy fields marked yellow and insert in UCSC browser “My Data”->”Custom Tracks”->”add custom tracks”.

Q1: List the elements (Vista IDs) that were tested for embryonic enhancer activity in the Gata4 TAD.

Q2: Which of these elements (in Q1) show cardiac enhancer activity at E11.5 (and in which compartments)?

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Task-3: Localize a mouse enhancer sequence in the mm10 genome and transfer a sequence into the H11 transgenic reporter framework.

In a 2004 study (https://pubmed.ncbi.nlm.nih.gov/15253934/) the lab of Brian Black (UCSF) discovered an AHF-enhancer sequence (Fig. 4).

-> Copy the mouse sequence and localize it in your UCSC browser session (using BLAT).

Right next to it you find the hs429 element (Vista Track) that was analyzed using random integration transgenic reporter analysis but no conclusive activity was found (negative).

-> Now you would like to reanalyze the hs429 element with a more accurate method (H11 transgenic reporter analysis).

For this you need to clone the element into an H11-compatible vector (PCR4-Shh::lacZ-H11). To do this, perform thefollowing steps:
1) Copy hs429 genomic sequence from UCSC browser (View -> DNA -> get DNA)

2) Upload the PCR4-Shh::lacZ-H11 plasmid sequence (available here: https://www.addgene.org/139098/) to a DNA modification tool (www.benchling.com)

3*) In Benchling (in silico), insert the hs429 sequence into the unique NotI side upstream of Shh minimal promoter sequence in a Gibson-assembly-compatible manner (helper tool here), by generating the Gibson primer sequences required.

 

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Task-4: Upload all H3K27ac heart datatracks from the ENCODE SCREEN database

Screen your locus of interest for signatures of promoters (PLS), proximal enhancers (pELS), distal enhancers (dELS), CTCF sites, accessible chromatin "only" or low accessible chromatin sites (see color code legend on slides or SCREEN website).
 

Instructions for upload of tracks from ENCODE Screen: e.g. heart tissue and craniofacial prominence

(automatically uploaded onto concurrent (or default) UCSC Browser session)

 

Go to SCREEN “overview”: https://screen.encodeproject.org

1) Select "Search Mouse (mm10, Registry V3)”

2) In the “cCRE” tab, select “Table View”

3) Select any "UCSC" button on the right (column: “genome browsers”)

4) Under "Available biosamples” search for your tissue type (or cell line) of interest, e.g. “heart” (C57BL/6 embryonic heart tissue) or “facial prominence” (C57BL/6 embryonic facial prominence).

5) Set a check mark for all samples of interest (all stages available, e.g. for heart: E10.5-E16.5, 8w, P0)

5) Click "Open in UCSC" (on top of the page). The tracks open directly in the UCSC Browser and should be added (as a track hub) to your most recent browser session

(required: unblock pop-up in browser settings for screen.wenglab.org)

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Task-5*: Identify which cardiac accessible chromatin regions (at E11.5) were already validated with transgenic reporter assays.

UCSC browser -> Tools -> Table Browser: select dataset, define region of interest, ”create” intersection, retrieve data as BED (output format)

Q1: Create a list of all Vista Enhancers overlapping a heart ATAC-seq peak.

Q2: List the elements (Vista IDs) that were tested for embryonic enhancer activity in the Tbx5 TAD (complex genomic landscape).
Q3: Create a list of heart ATAC-seq peaks with confirmed heart enhancer actifvity.