Methods

Question 1

What is Gro-Seq?

Answer 1

• Global run-on sequencing (GRO-seq) is the most widely used method to measure nascent RNA, and in recent years, it has been applied successfully to study the function and mechanism of action of noncoding RNAs.

Question 2

How does Gro-Seq work?

Answer 2

• GRO-seq captures the pieces of RNA that are being generated by active RNA polymerase. Think of it as if you were retrieving (parts of the) RNA molecule as it comes out of the copy machine that is RNA polymerase.
  
  • That means that most sequences will indeed correspond to gene regions (including introns, though!), but in contrast to normal poly(A)-enriched mRNA-sequencing you will mostly have captured incomplete transcripts.
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Question 3

What are some limitations of GRO-seq?

Answer 3

• Major limitations of GRO-seq are the laboriousness of the technique and the amount of starting material (the number of cells that are required lies in the 10⁷ range).

Question 4

What is Chromatin immunoprecipitation (ChIP)?

Answer 4

• Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell.
• It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly defining cistromes.

Question 5

What is the process of Chromatin immunoprecipitation (ChIP)?

Answer 5

The process in simple terms is as followed

1. DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP).
2. The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion.
3. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody.
   
   • Do this by bead-attached antibodies to immunoprecipitate target protein
4. The associated DNA fragments are purified and their sequence is determined. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo.

Question 6

What is Cross‐linking immunoprecipitation (CLIP)?

Answer 6

• CLIP (crosslinking immunoprecipitation) is a method used in molecular biology that combines UV cross-linking with immunoprecipitation in order to analyze protein interactions with RNA or to precisely locate RNA modifications

Question 7

What are the steps of CLIP?

Answer 7

1. CLIP begins with the in-vivo cross-linking of RNA-protein complexes using ultraviolet light (UV).
   
   • Upon UV exposure, covalent bonds are formed between proteins and nucleic acids that are in close proximity.
2. The cross-linked cells are then lysed, and the protein of interest is isolated via immunoprecipitation.
   
   • In order to allow for sequence specific priming of reverse transcription, RNA adapters are ligated to the 3’ ends, while radiolabeled phosphates are transferred to the 5’ ends of the RNA fragments.
3. The RNA-protein complexes are then separated from free RNA using gel electrophoresis and membrane transfer.
4. Proteinase K digestion is then performed in order to remove protein from the RNA-protein complexes.
   
   • This step leaves a peptide at the cross-link site, allowing for the identification of the cross-linked nucleotide.
5. After ligating RNA linkers to the RNA 5’ ends, cDNA is synthesized via RT-PCR.
6. High-throughput sequencing is then used to generate reads containing distinct barcodes that identify the last cDNA nucleotide.
   
   • Interaction sites can be identified by mapping the reads back to the transcriptome.

Question 8

How do you purify biotinylated proteins?

Answer 8

Question 9

What is EMSA?

Answer 9

• EMSA is a common affinity electrophoresis technique used to study protein–DNA or protein–RNA interactions.
• This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex.
• Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation, DNA replication, DNA repair or RNA processing and maturation, as well as pre-mRNA splicing.

Question 10

Describe this EMSA

Answer 10

• Lane 1 is a negative control, and contains only genetic material.
• Lane 2 contains protein as well as a DNA fragment that, based on its sequence, does not interact.
• Lane 3 contains protein and a DNA fragment that does react; the resulting complex is larger, heavier, and slower-moving.
• The pattern shown in lane 3 is the one that would result if all the DNA were bound and no dissociation of complex occurred during electrophoresis.
• When these conditions are not met a second band might be seen in lane 3 reflecting the presence of free DNA or the dissociation of the DNA-protein complex.

Question 11

What is an EMSA supershift?

Answer 11

• An antibody that recognizes the protein can be added to the SNA-Protein mixture to create an even larger complex with a greater shift.
• This method is referred to as a supershift assay and is used to unambiguously identify a protein present in the protein-nucleic acid complex.

Question 12

What is rtPCR?

Answer 12

• It is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).
• It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
• Real-time PCR can be used quantitatively (quantitative real-time PCR), and semi-quantitatively, i.e. above/below a certain amount of DNA molecules (semi quantitative real-time PCR).

Question 13

What is ChIP-sequencing?

Answer 13

• ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA.
• ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.
• It can be used to map global binding sites precisely for any protein of interest.

Question 14

What are the steps of ChIP-seq?

Answer 14

• ChIP-seq protocols have been adapted from ChIP-chip methods: proteins are cross-linked to their bound DNA by formaldehyde treatment, cells are homogenized, and chromatin is sheared and immunoprecipitated with antibody-bound magnetic beads.
• The immunoprecipitated DNA is then used as the input for a next-generation sequencing library prep protocol, where it is sequenced and analyzed for DNA binding sites.
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Question 15

Wha is CLIP-seq?

Answer 15

• CLIP-Seq combines UV crosslinking and immunoprecipitation with high-throughput sequencing to identify binding sites of RNA-binding proteins.
• CLIP-seq depends on cross-linking induced mutation sites (CIMS) to localized protein-RNA binding sites.[4] Because CIMS are reproducible, high sequencing depths allow CIMS to be differentiated from technical errors.

Question 16

What is a Tethering experiment?

Question 17

Describe the process of CLIP

Question 18

Describe the process of CHIP

Answer 18

Question 19

Describe the process of Gro-Seq?

Answer 19

Question 20

What is heat map analysis?