TITLE: Single-cell Epi2-Seq
AUTHORS: Christoph  Geisenberger, Jeroen van den Berg, Vincent van Batenburg, Buys De Barbanson, Jeroen de Ridder, Alexander van Oudenaarden

[DESCRIPTION]
Here we describe the full protocol for single-cell Epi2-Seq which enables joint readout of histone modifications and DNA methylation in individual cells. In short, this methods combines antibody-targeted MNase digestion (ChIC, CUT&RUN) with Tet-Assisted Pyridine Borane Sequencing (TAPS), a bisulfite-free technique for base-resolution DNA methylation profiling. TAPS, unlike bisulfite, specifically converts 5mC which increases mapping rates and makes it feasible to use unmethylated single-cell adapters. Also, DNA damage is minimal which reduces input requirements and makes it well suited for single-cell applications. 

[STEPS]
SECTION: Preparation of Tet1 Reaction Buffer and Fe2+ solution
5. Assemble TET1 reaction buffer (volumes are suggestions and can be scaled up or down)

 

 

SECTION: Chromatin Immuno-Cleavage (ChIC)
10. Fixation and permeabilization
Harvest cells and wash twice with PBS at room temperature (centrifuge 3 min, 500 g to pellet)
Resuspend cells in 300 µl PBS per 106 cells on ice
Add 700 µl of ice-cold absolute ethanol per 106 cells while vortexing gently (70% ethanol final)
Cells are fixed for two hours at -20°C
Wash cells twice with WB1 (see above)
Resuspend cells in 500 µl WB1
Transfer reaction to 0.5 ml protein lo-bind Eppendorf tubes

 

SECTION: FACS
14. Prepare sorting plates
Add 10 µl of sterile filtered mineral oil to each well of 384-well hard-shell plates
Plates can be prepared in advance, sealed and kept for multiple months

Cell sorting
Cells were sorted on a BD InfluxTM cell sorter
Depending on the machine and application, Hoechst signal can be used to select cells in G1 phase and to avoid debris
After sorting, centrifuge plates for 1 min at 2,000 g


 


SECTION: Plate pooling
20. Remove cover, attach 384-well plates upside-down to a VBLOK200 Reservoir
Cover with parafilm
Centrifuge 2 min @ 500 g at room temperature to collect liquid in reservoirs
Transfer aqueous phase to fresh 1.5 ml DNA lo-bind Eppendorf tube
Centrifuge 1 min @ 13,000 g, transfer aqueous phase to fresh tube
Repeat centrifugation and transfer once
Measure volume with pipette
Perform Ampure XP SPRI bead clean up (bead-to-sample ratio = 0.8)
resuspend in 19 µl nuclease-free water
transfer sample to fresh 0.5 ml DNA lo-bind Eppendorf tube
SECTION: In-vitro transcription (IVT)
24. Assemble IVT reaction (all reagents are part of MegaScriptTM T7 transcription kit):

 
Incubate 14 hours @ 37°C (with lid set to 70°C)
To each reaction, add 6 µl of nuclease-free H2O and Turbo DNAse (part of MegaScriptTM T7 transcription kit)
Incubate 15 min @ 37°C
Add 7.88 µl of RNA fragmentation Buffer (200 mM Tris-Acetate, 500 mM KaOAc, 150 mM MgOAc)
Incubate samples 90s at 94°C, immediately chill on ice
Add 4.13 µl of 0.5 M EDTA to capture Mg2+
Clean up samples with 34 µl (0.8x) of RNAClean XP beads
Elute in 6 µl of nuclease-free H2O

 

  

SECTION: Preparation of sequencing libraries
25. In a 0.5 ml DNA lo-bind tube, combine: 
5 µl aRNA
0.5 µl of 10 mM dNTP solution
1 µl random hexamer RT primer 20 µM 
SECTION: Preparation of Tet1 Reaction Buffer and Fe2+ solution
4. Prepare 0.5 M Fe2+ Solution
Dissolve 1.96 g of Iron(II) sulfate hexahydrate in 10 ml nuclease-free H2O
Store Fe2+ solution in single-use aliquots at -80°C
SECTION: Preparation of Tet1 Reaction Buffer and Fe2+ solution
3. Prepare 1 M solutions of Ascorbic Acid and a-Ketoglutarate:
a-Ketoglutarate: Dissolve 0.9503 g in 5 ml nuclease-free H2O
Ascorbic Acid: Dissolve 0.8806 g in 5 ml nuclease-free H2O

 

SECTION: TAPS conversion
21. Assemble the following reaction on ice:

 


 


Incubate for 80 min @ 37°C
Add 1 µl of Proteinase K
Vortex and centrifuge briefly to collect liquid
Incubate 15 min @ 55°C
Perform a 2x Ampure XP SPRI cleanup 
Option 1: to repeat Tet1 oxidation, elute in 20 µl nuclease-free water and repeat above reaction
Option 2: to continue to Pyridine Borane incubation, elute in 33.75 µl nuclease-free H20 and transfer volume to a fresh 1.5 ml Eppendorf tube
SECTION: TAPS conversion
22. 
 


Assemble Pyridine Borane reaction at room temperature:

 

Incubate 16 hours @ 37°C in thermal shaker set to 850 rpm

 

SECTION: TAPS conversion
23. Use Zymo Oligo Clean & Concentrator kit to clean up reactions after pyridine borane incubation
Elute DNA with 15 µl of nuclease-free H2O heated 60°C
To maximize DNA retrieval, repeat elution once (final volume ~ 30 µl)
Reduce volume to 9.6 µl
Option 1: Incubate in speed-vac, check volume regularly to prevent over-drying
Option 2: Peform 1x Ampure XP SPRI clean-up, elute in 9.6 µl nuclease-free H2O
SECTION: Chromatin Immuno-Cleavage (ChIC)
9. Recipes for wash buffers used in Chromatin Immuno-cleavage (ChIC)

 

SECTION: Chromatin Immuno-Cleavage (ChIC)
11. Incubation with primary antibody
Add histone-specific antibody (see table below for antibodies used in publication, others need to be titrated)
Incubate cells overnight at 4°C with gentle agitation (e.g. on a roller)

 

SECTION: Chromatin Immuno-Cleavage (ChIC)
12. pA-MNase binding and nuclear staining
Wash cells once with 500 µl WB2
Add pA-MNase to a final concentration of 3 ng/µl
Add Hoechst 34580 to a final concentration of of 5 µg/ml
Incubate 1 hour at 4°C with gentle agitation
SECTION: PA-MNase production
1. (Single-cell) Chromatin immunocleave requires expression of a pA-MNase fusion protein in bacteria. We refer to Zeller et al. (Nature, 2022) and Schmid et al. (Mol. Cell, 2004) for detailed protocols (see references for more information). However, pA-MNase is also available commercially through Cell Signaling Technology (cat. no 4036).
SECTION: Chromatin Immuno-Cleavage (ChIC)
13. Washing and straining
Wash cells twice with WB2
Resuspend cells in 500 µl of WB3
Filter cells through a 70 µM strainer
Transfer to FACS tubes
SECTION: Single-cell processing
16. MNase digestion and Protease K digest
MNase digestion is initiated by dispensing 100 nl per well of WB3 supplemented with 2 mM CaCl2 per well
Incubate 30 mins in thermocycler set to 4°C
to stop digestion, dispense 100 nl per well of the mix below
Incubate: 20 min @ 4°C -> 6 hrs @ 65°C -> 20 min @ 80°C -> hold at 4°C

 

SECTION: Single-cell processing
15. General notes:
Nanoliter dispension was performed with the Innovadyne Nanodrop II platform. However, euqivalent nanoliter dispenser such as the iDOT or Mantis can be used aswell
Adapters were copied from a source plate using the TTP Labtech Mosquito HTS liquid handler

 

SECTION: Single-cell processing
17. Fragment blunting
Dispense 150 nl per well of the mix below (350 nl total at this point)
Incubate: 30 min @ 37°C -> 20 min @ 75°C -> hold @ 4°C

 

SECTION: Single-cell processing
18. A-tailing
Dispense 150 nl per well of the mix below (500 nl total at this point)
Incubate: 15 min @ 37°C -> 10 min @ 72°C -> hold @ 4°C

 

SECTION: Single-cell processing
19. Addition of adapters and ligation
per well, add 50 nl of adapters from source plate (5 µM) using the Mosquito HTS
dispense 150 nl of ligation mix per well (700 nl total at this point)
Incubate: 20 min @ 4°C -> 16 hrs @ 16°C -> 10 min @ 65°C -> hold @ 4°C


 

SECTION: mTet1 Protein production
2. Tet-assissted bisulfite sequencing (TAPS) requires expression of the catalytic domain of mouse Tet1 (mTet1CD) in an eukaryotic expression system. We refer to the detailed protocol by Liu et al. (Nature Biotechnology, 2019) for more information. mTet1CD can also be purchased from Sigma Aldrich (S9797-25UG).
SECTION: Preparation of sequencing libraries
26. Heat samples to 65°C for 5 minutes
Immediately chill samples on ice
Assemble the reaction below
Incubate: 10 min @ 25°C -> 60 min @ 42°C -> hold @ 4°C

 

SECTION: Preparation of sequencing libraries
27. Assemble the reaction below
Incubate: 30 s @ 98°C -> 10 - 13 x (10 s @ 98°C, 30 s @ 60°C, 30 s @ 72°C) -> 10 min @ 72°C -> hold @ 4°C
Perform two AMPure XP SPRI bead cleanups (bead-to-sample-ratio 0.8)
Elute amplified sequencing library in 15 µl of nuclease-free H2O

 

 

SECTION: Preparation of sequencing libraries
28. Sequencing library QC
Measure concentration with a dye-based method such as Qubit
Run 1 µl of library on Agilent High Sensitivity Bioanalzyer to assess size distribution
Perform Illumina sequencing according to manufacturers protocol 
SECTION: DNA sequences
29. Barcoded single-cell Adapters
Example Top strand
5’-GGTGATGCCGGTAATACGACTCACTATAGGGAGTTCTACAGTCCGACGATCNNNACACACTAT
Example Bottom strand
5’-/5Phos/TAGTGTGTNNNGATCGTCGGACTGTAGAACTCCCTATAGTGAGTCGTATTACCGGCGAGCTT

Sequence features: Fork -> T7 promoter (bold) -> RA5 Illumina sequence -> 3 bp UMI (NNN) -> single-cell barcode (bold & italic) -> single-base T overhang

Library Amplification
RandomhexamerRT primer
GCCTTGGCACCCGAGAATTCCANNNNNN
Barcoded RPIX primer
5’-CAAGCAGAAGACGGCATACGAGAT-[6bp]-GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RP1 primer
5’-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA



 

SECTION: Preparation of methylated lambda phage spike-ins
6. Methylation of lambda phage DNA
Assemble the reaction below on ice
Incubate: 2 hours @ 37°C
Add an additional 1 µl of SAM and 0.5 µl M.SssI
Incubate: 2 hours @ 37°C
Perform Ampure XP SPRI cleanup (bead-to-sample ratio = 1:1)
Elute sample in 20 µl of nuclease-free H20
Repeat reaction once with methylated sample as input (including top-up of SAM)
Perform Ampure XP SPRI cleanup (bead-to-sample ratio = 1:1)
Elute sample 20 µl of nuclease-free H20

 
* amount has to be adjusted based on batch-dependent concentration
SECTION: Preparation of methylated lambda phage spike-ins
7. NlaIII digestion
Assemble the reaction below on ice
Note: Adapter should be added in a ratio of 10:1, calculate molarity based on Qubit measurement and assuming full NlaIII digestion 
Incubate: 2 hours @ 37°C -> 20 min @ 65°C -> hold @ 4°C
Perform Ampure XP SPRI cleanup (bead-to-sample ratio = 1:1)
Elute sample 20 µl of nuclease-free H20
Measure concentration with dye-based method (e.g. Qubit)

 

SECTION: Preparation of methylated lambda phage spike-ins
8. Adapter ligation
Assemble the reaction below on ice
Note: Adapter should be added in a ratio of 10:1, calculate molarity based on Qubit measurement and assuming full NlaIII digestion 
Incubate: 20 min @ 20°C -> 10 min @ 65°C -> hold @ 4°C
Perform 2 Ampure XP SPRI cleanups (bead-to-sample ratio 0.8:1)
Elute fully methylated, adapter-ligated controls in nuclease-free water
Prepare aliquot with a concentration of 7 pg/µl