✂️ & 🏷️ R-loop

Intro

CUT&Tag Resource Center
CUT&Tag-IT® R-loop Service
CUT&Tag-IT® R-loop Assay Kit
CUT&Tag-IT® Spike-In Control, R-loop

1. Cut & Tag protocol

Note
we used reduced volumes of reagents/solvents when we weren’t sure if we had enough (specified in Marketa’s protocol)

PDF: Protocol; CUT&Tag-IT™ R-loop Assay Kit
PDF: CUT&Tag-IT® Spike-In Control, R-loop
PDF: Counting Cells and Nuclei for Epigenetic Applications

Library Preparation for Illumina

• I find this one very usefull
• it includes also videos with “real life tips” on right side - description of each bellow
  

Library Preparation for Illumina: text & videos

2. Concentration

Qubit & Tapestation
For a professional NGS library prep, the standard protocol:
Use the TapeStation for the “Size” and the Qubit for the “Concentration.”

• When pooling libraries for an Illumina run, even a small error in concentration can lead to “index hopping” or uneven data distribution (where one sample gets 90% of the reads and others get 10%).
• Using the TapeStation to check size and the Qubit to check molarity ensures your final calculations for pooling (converting ng/µL to nM) are as accurate as possible.
  
  When can you skip one?
  If you are in a situation where you must choose just one to save time or sample:
• TapeStation only : samples are all the exact same type (e.g., all the same PCR amplicon length) and you have a very high concentration.
• Qubit only: already know exactly what size your fragments are (e.g., a specific 300bp PCR product) and don’t need to check for degradation.
  (source: Gemini)

2A. Qubit

Note

• protocols are usualy everywhere with Qubit
• there are special tubes for it
  
  Qubit dsDNA Quantification Assay Kits
  1. dsDNA
  2. dsDNA: High Sensitivity
  3. lower and upper point calibration (S1 and S2)
  4. dilution of samples 1:200 : 1 μL + 199 μL
  5. units: ng/μL
     
     Workflow
• samples on ice
• thaw at r.t. (but keep on the ice rest of the time)
• “flick the samples” – to homogenize
• spin down (short spin at r.t. centrifuge)
• mix with buffer (pipet first buffer 199μL and then sample 1μL)
• quick vortex
• incubation (r.t.; 3 min; can differ base of kit)
• measure (ng/μL) + write down a concentrations
• optional: download data on USB
  
  Qubit™ 4 Fluorometer: pg 18-28
  

Calculation of dsDNA library concentration

• if you would like to calculate concentration from Qubit
• dsDNA library concentration
  
  

2B. Tapestation

Our video protocol for Agilent TapeStation Software 5.1

• Agilent High Sensitivity D1000 ScreenTape Assay Quick Guide for 4200 TapeStation System
  
• You Toube: TapeStation tutorial - very nicely explained; Workflow: sapmple preparation from 01:00; Workflow: TapeStation from 08:27; Workflow: Software from 09:37
  
• You Toube: Agilent TapeStation: DNA and RNA Applications; Workflow from 14:54
  
• Agilent TapeStation
  

3. Pooling

• Pooling Calculator Illumina
  

• concentration from libraries
• elution buffer as diluent for pooling - correct concentration of Tris
  

Novaseq X

• pool samples equimolarly
• the minimum 5nM to guarantee the best results
• 35 μL per lane (minimum volume); can be higher
• pool in a 1.5 mL low-bind tube

Under-represented samples
Example

• pool all experimental samples equimolarly (5nM)
• pool the 4 negative controls at a slightly lower molarity
  

It is better to discuss dilution with the genomics center before pooling.

Let them know, which assay was used at TapeStation - for their quality assesment (HS D1000, or they can try D5000 for longer fragments).

Further resources

NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®

INSTRUCTION MANUAL

Multiplex Oligos for Illumina Protocol

Click

Learn about Illumina’s Next-Generation Sequencing Workflow

Illumina’s Next-Generation Sequencing Workflow

Calculation of dsDNA library concentration

• Click