Biochemical and analytical protocols

• Homemade EU click
  • Stock solutions
  • Add EU to cells
  • Dissociate
  • Fix cells
  • Permeabilize
  • Prepare click/label mix
  • Label and wash
• HIVE scRNA-seq
  • Sample capture
  • HIVE processing
• Immunofluorescent Staining
  • Solutions Required
  • Adherent Cell Staining
  • Cell Staining for Flow
• ATAC-see
  • Transposase buffer preparation
  • ATAC-see
• CellBaum User’s Guide
  • Local Usage
  • MIT Supercloud Usage
• CellTrace Dye Labeling
  • CellTrace dyes available in lab
  • Other materials needed:
• Sample Prep and Confocal Imaging at the Koch Microscopy core
  • Confocal access and Experimental planning:
  • Confocal imaging
  • Saving and Export of files
• HCR DNA-FISH
  • Fixation
  • Hybridization
  • Probe Preperation
  • Post-Hybridization
• Halo-tag Labeling
  • Halo-tag cell permeable dyes available in lab
  • Other materials needed:
• HCR RNA-FISH
  • Day 1
  • Day 2
  • Day 3
• Live nuclear staining using Hoechst
  • Materials
  • Protocol
• Image segmentation and particle analysis in ImageJ
  • Defining regions of interest (ROI)
  • Quantifying nuclear-localized foci
  • Merging images in ImageJ
• KAPA SYBR® FAST qPCR
  • Protocol
  • 1. Master Mix Preparation
  • 2. Reaction Setup
  • 3. qPCR
  • 4. Data Analysis
• Image analysis using the KI workstation - Nemo
  • LasX 3D Image Viewer
  • Imaris
• Magnetic bead clean-up for genomics libraries
  • Uses
  • Determine Bead Volume to use by size range you wish to recover:
  • Preparations:
  • Basic Bead Clean Protocol:
  • 2-step “Big” bead clean to remove large fragments >~350bp.
• Nuclei Isolation for Downstream Genomics Applications
  • Nuclear Isolation
  • Recipes
• Psoralen-qPCR supercoiling assay
  • Experiment setup
• sci-ATAC-seq
  • Background
  • Preparation (N days before)
  • Day 1: cell collection and fixation
  • Day 1: transposition
  • Day 2: library amplification
  • Day 3: library quantification
• Single cell qPCR
  • Single-cell acquisition
  • Lysis, reverse transcription (RT), and specific target amplification (STA)
  • Single-cell qPCR on Fluidigm Biomark
• SingleShot RNA isolation and Reverse Transcription
  • Materials need for RNA isolation
  • Materials needed for cDNA synthesis
  • RNA isolation using SingleShot Cell Lysis
• Snap-tag Labeling
  • Snap-tag cell permeable dyes available in lab
  • Other materials needed:
• Titration of Tn5 transposition in MEFs
  • Part 1. Assembling the Transposase
  • Part 1.1 Prepping the Tn5 concentrations from Assembled Tn5 transposome per Transposition reaction (50uL)
  • Part 2. Transposition Reaction of Isolated nuclei
• Western Blot
  • Lysis
  • Bradford Assay
  • Protein Gel Casting
  • Loading and Running the Gel
  • Coomassie Staining
  • Transferring the protein from the gel to the membrane
  • Antibody Staining