RNA Detection

Place the tube containing lysate and the beads on the magnet and transfer the supernatant to a prechilled new tube on ice. 3.4 ...

Place the frozen tissues into the Dounce homogenizers con- taining 400μL of ice-cold RO-lysis buffer (10 w/v % tissues in RO-ly ...

If genotyping is needed, prepare tail or toe biopsies for geno- mic DNA PCR experiments. Clean the equipment to avoid carry-ove ...

Because the efficiency of the lysis in this condition is signifi- cantly lower than in the condition using detergents and cyclo ...

References Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M (2011) Global quantification of ...

Chapter 6 LCM-Seq: A Method for Spatial Transcriptomic Profiling Using Laser Capture Microdissection Coupled with PolyA-Based RN ...

sequencing of neurons isolated from mouse and human postmor- tem tissues down to single cells. We utilized LCM as it enables pre ...

Embedding medium (OCT). Embedding molds. Block of dry ice. Forceps, precooled on dry ice. Razor blade or scalpel to trim tissue ...

2.4 Antibody Based Rapid Tissue Staining Aluminum foil to cover light-sensitive staining solutions. 15 mL centrifuge tubes. Sta ...

oligo-dT: 50 AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTT- TTTTTTTTTTTTTTTTTTTVN3^0 (HPLC purified, store at 10 μM concentration in ddH ...

Master mix 5 (Index ligation and enrichment PCR): 10μL5 high fidelity PCR buffer, 1.5μL dNTPs, 1μL high fidelity PCR enzyme, a ...

3.3 Histological Tissue Staining for Laser Capture Microdissection Transport slides (in slide boxes) on dry ice. Use forceps to ...

Thaw slides for 30 s at room temperature by placing horizon- tally on a clean surface (e.g., 5 mL pipette tips on plastic tray) ...

Before initiating the staining, switch on the LCM system (microscope, laser, computer, start LMD software) and turn on the UV l ...

Place 0.2 mL PCR tubes into the collector and check tubes for obvious contamination (change if contaminated) (seeFig. 3). Place ...

3.6.1 Reverse Transcription and PCR Amplification Preheat thermocycler to 72C. Prepare master mix 1 and 2 and place the metal ...

Leave the plate without cover to air-dry the beads. Dry bead pellets will have tiny cracks (seeNote 18). Add 17μL EB; flush tow ...

3.6.3 Tagmentation and Sequencing Index Ligation Preheat the PCR machine to 55C. Prepare the tagmentation master mix 4 on ice, ...

Spinal cord dissections take a maximum of 15 min from decap- itating the animal and brains can be retrieved in less than 3 min. ...

The secondary antibody should be of high quality and used at a high concentration to minimize incubation times and thus unneces ...