Jul 14, 2025
Fiber-seq for flash frozen mouse tissue for IGVF
- Jasmine Sakr1
- 1UCI
Protocol Citation: Jasmine Sakr 2025. Fiber-seq for flash frozen mouse tissue for IGVF. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62qpkgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 05, 2024
Last Modified: July 14, 2025
Protocol Integer ID: 109200
Keywords: Fiber-seq, open chromatin, methylation, m6A, methyltransferase, cortex, mouse, mouse brain, IGVF, UCI, whole genome sequencing, DNA, genome, Mortazavi, nuclei isolation, seq for flash frozen mouse tissue, seq experiment for frozen mouse tissue, hmw dna extraction kit for tissue, nuclei from flash frozen mouse, flash frozen mouse tissue, frozen mouse tissue, dna extraction, hmw dna extraction kit, nuclei extraction, frozen lcl cell, hippocampus for igvf, flash frozen mouse, hprc fiber, igvf this protocol, fiber, seq experiment, seq protocol, dna, seq protocol on dmso, tissue, igvf, seq
Funders Acknowledgements:
NHGRI
Grant ID: HG012077
Abstract
This protocol describes Fiber-seq experiment for frozen mouse tissue and has been optimized from the following protocols:
Nuclei extraction: Protocol to isolate and fix nuclei from flash frozen mouse left cortex and hippocampus for IGVF, Version 2.1 (January 2023)
Labelling: HPRC Fiber-seq protocol on DMSO frozen LCL cells, (March 2024)
DNA extraction: Monarch‱ HMW DNA Extraction Kit for Tissue (NEB #T3060S/L), Version: 2.1_4/21
Guidelines
Try working quickly to help maintain nuclei integrity and prevent over labelling.
Materials
Reagents
| Name | Manufacturer | Cat # | |
| PBS | HyClone | SH30256 | |
| 7.5% BSA | Life Technologies | 15260037 | |
| Monarch® HMW DNA Extraction Kit for Tissue | New England Biolabs | T3060 | |
| EcoGII Methyltransferase | New England Biolabs | M0603S | |
| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | |
| Spermidine | Thermo Scientific | A19096 | |
| SDS, 20% Solution | Thermo Scientific | AM9820 |
Supples and Equipment
| Name | Manufacturer | Cat # | |
| gentleMACS™ C Tube | Miltenyi Biotec | 130-093-237 | |
| gentleMACS™ Octo Dissociator | Miltenyi Biotec | 130-095-937 | |
| gentleMACS™ Octo Coolers | Miltenyi Biotec | 130-130-533 | |
| MACS SmartStrainers (70 um) | Miltenyi Biotec | 130-110-916 | |
| MACS SmartStrainers (30 um) | Miltenyi Biotec | 130-098-458 | |
| Eppendorf ThermoMixer® C | Eppendorf | - | |
| Eppendorf SmartBlock 2.0 mL | Eppendorf | 5362000035 |
Troubleshooting
Set Up
Make buffers and solutions
- Buffer A
| Final Concentration | Stock Concentration | Volume stock soln | |
| Nuclease-free H2O | 100% | 95.8 mL | |
| 15 mM Tris-Cl, pH 8.0 | 1 M Tris-Cl, pH 8.0 | 1.5 mL | |
| 15 mM NaCl | 5 M NaCl | 300 μL | |
| 60 mM KCl | 3 M KCl | 2 mL | |
| 1 mM EDTA, pH 8.0 | 0.5 M EDTA, pH 8.0 | 200 μL | |
| 0.5 mM EGTA, pH 8.0 | 0.5 M EGTA, pH 8.0 | 100 μL |
This buffer is used in the labelling reaction and is table at room temperature.
- 0.5 Molarity (M) Spermidine and store at 4 °C
- 20 % (v/v) Sodium Dodecyl Sulfate (SDS) and store at Room temperature
Pre-chill centrifuge to 4 °C
Set two thermomixers at 56 °C and 25 °C
Prepare and label the following tubes for DNA extraction (for each labeling reaction):
- Bead strainer in a collection tube
- 2 mL tube for wash step
- 2 mL tube for elution step
- 1.5 tube for final elution
Add 150 µL of gDNA Elution Buffer II to the 2 mL elution tubes
Prepare an ice bucket and keep the following on ice
- Proteinase k and RNAse A
- Pre-chill gentleMACS C Tube and two 5 mL tubes
- 0.5 Molarity (M) Spermidine
Nuclei Extraction
Make enough RSB for the total number of samples.
| Reagent | Final conc. | 1 | 2 | 3 | 4 | |
| PBS | 3.45 mL | 6.41 mL | 9.37 mL | 12.33 mL | ||
| 7.5% BSA | 0.1% | 46.67 µL | 86.67 µL | 126.67 µL | 166.67 µL |
Or a large stock can be stored at 4ºC for a month.
Keep flash frozen tissue samples on dry ice until lysis.
Add 2 mL Nuclei Extraction Buffer to a chilled gentleMACS C Tube.
Drop whole frozen tissue into the chilled gentleMACS C Tube with 2 mL Nuclei Extraction Buffer.
Invert immediately, ensuring tissue is not stuck to the bottom or side. Proceed immediately to load onto Octo Dissociator and add Octo Cooler over each gentleMACS C Tube.
Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (00:05:00 ).
5m
Remove the gentleMACS C Tube and ensure tissue did not get stuck on the sides by tapping the tube.
Spin down in4 °C centrifuge for 00:00:10 .
10s
Mix the nuclei suspension thoroughly by pipetting with a 5 mL pipet.
Filter nuclei suspension through 70 µm MACS SmartStrainer into a 5 mL tube.
Wash empty, used gentleMACS C Tube with an additional 2 mL Nuclei Extraction Buffer. Close the cap and swish to recover any nuclei stuck to the sides.
Filter the 2 mL additional Nuclei Extraction Buffer through the 70 µm MACS SmartStrainer into the same 5 mL tube (total volume now 4 mL).
Centrifuge the 4 mL nuclei suspension at 350 x g, 4°C, 00:05:00 .
5m
Remove the supernatant without disrupting the pellet.
Resuspend nuclei pellet in 3 mL RSB.
Filter nuclei suspension through 30 µm MACS SmartStrainer into a new 5 mL tube.
Make aliquots of 5 million nuclei in 1.5 mL tubes.
Labelling
Spin 5 million nuclei at 250 x g, 4°C, 00:05:00 .
5m
During the 5 min, mix the following and keep Reaction Master Mix On ice
Buffer A + Spermidine Room temperature
| Number of tubes | 1 | 2 | 3 | 4 | |
| Buffer A | 500 μL | 750 μL | 1 mL | 2 mL | |
| 0.5 M Spermidine | 0.5 μL | 0.75 μL | 1 μL | 2 μL |
This is made fresh on the day of the experiment.
Reaction Master Mix (75 U) On ice
| Number of tubes (+0.2 extra) | 1 | 2 | 3 | 4 | |
| Buffer A + Spermidine | 277.5 µL | 610.5 µL | 888 µL | 1,165.5 µL | |
| EcoGII Methyltransferase (200 U) | 15 µL | 33 µL | 48 µL | 63 µL | |
| 32 mM SAM | 7.5 µL | 16.5 µL | 24 µL | 31.5 µL |
This reaction uses 75 units of enzyme and 160 µM SAM with a total reaction volume of 300 µL.
Remove supernatant and resuspend the pellet in 300 µL of Reaction Master Mix buffer.
Incubate on 25 °C heat block for 00:10:00 .
10m
Add 15 µL of 20% SDS to each sample (final concentration of 1%)
Thoroughly mix by gently inverting tube. Do not vortex or pipette to mix.
DNA extraction
10m
Add20 µL Proteinase k and 10 µL RNAse A to each tube
Mix gently by pipetting using wide-bore tip.
Incubate in a thermal mixer at 2000 rpm, 56°C, 00:10:00 .
10m
Add 300 µL Protein Separation solution.
Mix on vertical rotating mixer for 20 rpm, 00:01:00 .
1m
Centrifuge for 16000 x g, 00:10:00 .
10m
Using a wide-bore tip, transfer as much of the upper phase containing the DNA to a labeled, empty 2 mL tube without disrupting the pellet at the bottom.
Add 2 DNA capture beads to each sample.
Add 425 µL of isopropanol.
Mix on a vertical rotating mixer at 10 rpm, 00:10:00 to attach DNA to the beads.
During the wash steps, work quickly to prevent DNA from drying out on beads!
Discard liquid by keeping tube upright, insert pipette tip and gently push beads aside to remove liquid.
Add 500 µL Wash Buffer.
Mix by gently inverting the tube 3 times.
Discard liquid by keeping tube upright, insert pipette tip and gently push beads aside to remove liquid.
Add 500 µL Wash Buffer.
Mix by gently inverting the tube 3 times.
Discard liquid by keeping tube upright, insert pipette tip and gently push beads aside to remove liquid.
Pour the beads into the bead retainer that is in a collection tube.
Pulse spin (≤ 1 second) the tubes to remove any residual wash buffer from the beads.
Separate the bead retainer from the collection tube and pour the beads into the labeled tube with 150 μL of elution buffer and save the bead retainer for a future step.
Incubate on thermal mixer at 56 °C for 00:10:00 and during incubation, make sure all sides of the beads are exposed to elution buffer by tilting the tube almost horizontally and gently shaking after the first 5 min.
10m
Incubate at Room temperature for 01:00:00 or 4 °C Overnight for the DNA to homogenize .
1h 10m
Pour the eluate and the glass beads into the bead retainer that is placed in the final labeled tube.
Centrifuge for 12000 rpm, 00:00:30 to separate the eluate from the glass beads.
30s
Mix gently by pipetting using wide-bore tip.
Qubit each tubes and keep on ice until library prep or store in -20°C
Library building
Follow "ONT Library Prep for Whole Genome Sequencing" protocol to build libraries for whole genome sequencing using Oxford Nanopore platform.