Journal: Nature protocols

Article Title: Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting.

doi: 10.1038/s41596-021-00630-1

Figure Lengend Snippet: Fig. 1 | Overview of the experimental workflow. a, Nuclei are extracted using a hypotonic buffer. Methylation footprinting is performed by incubating the nuclei with a GpC (M.CviPI) and, optionally, CpG (M.SssI) methyltransferase (Mtase). Regions accessible to the enzymes are methylated, while regions bound by proteins (TFs, nucleosomes) are protected, creating footprints of various sizes. DNA is extracted and used for whole-genome (left) or targeted amplicon (right) analysis. b, For whole-genome analysis, DNA is fragmented to a target size range of 300–500 bp. DNA is end- repaired, and sequencing adapters are ligated. An optional capture step can be performed to enrich the library for regions of interest such as CREs and reduce the sequencing depth required for single-molecule analysis. c, DNA is bisulfite converted and the library is amplified before sequencing on Illumina MiSeq and NextSeq platforms. d, Alternatively to the whole-genome approach, primers can be designed to target 96 loci using amplicon bisulfite PCR. Amplicons are typically designed to cover 300–500 bp of the CRE. e, Amplicons are pooled, and the library is prepared. Up to 12 libraries can be multiplexed and sequenced on a MiSeq instrument. The read ends in amplicon data are identical for every molecule, creating focused high-coverage views of the targeted loci.

Article Snippet: ● Bioanalyzer 2100 instrument (Agilent, cat. no. G2939BA) ● Bioanalyzer DNA 1000 Kit (Agilent, cat. no. 5067-1504) ● Bioanalyzer High Sensitivity DNA Kit (Agilent, cat. no. 5067-4627) ● Centrifuge, refrigerated, with fixed-angle rotor (Eppendorf, model no. 5427R) ● Centrifuge with fixed-angle rotor (Eppendorf, model no. 5425) ● Centrifuge with swinging bucket (Eppendorf, model no. 5810R) ● Heater block with wells for 1.5 ml tubes (e.g., Thermo), set to 37 and 56 °C ● Magnetic rack for PCR tubes ● Magnetic rack for 1.5 ml tubes; Dynamag (Thermo, cat. no. 12321D) ● Microcentrifuge (e.g., Roth) ● 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 22-282) ● 1.5 ml microcentrifuge safe-lock tubes (Eppendorf, cat. no. 30120086) ● 1.5 ml microcentrifuge DNA LoBind tubes (Eppendorf, cat. no. 30108051) ● 0.2 ml PCR tubes (Eppendorf, cat. no. 30124359) ● Thermal cycler (Biorad, C1000 touch, cat. no. 1851148/1851196) ● Vortex mixer (e.g., Vortex Genie; VWR) ● Water baths (e.g., VWR), set to 37 °C 5684 NATURE PROTOCOLS | VOL 16 |DECEMBER 2021 | 5673–5706 |www.nature.com/nprot

Techniques: Methylation, Footprinting, Amplification, Sequencing

Journal: Nature protocols

Article Title: Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting.

doi: 10.1038/s41596-021-00630-1

Figure Lengend Snippet: Fig. 9 | QCs during the preparation of amplicon SMF samples. 1–2 µg of footprinted DNA is bisulfite converted and used as an input for 96 parallel PCR reactions. a, PCR efficiency is checked by loading an aliquot on a 2% agarose gel. With standard bisulfite primer design parameters, 80–90% of the reactions lead to a detectable product and amplicon size ranges between 300 and 500 bp (Step 137). An aliquot of each PCR product is pooled and used as an input for sequencing library preparation. b, The size distribution of the final library is verified on an Agilent Bioanalyzer, with an expected size of 430–630 bp (Step 176).

Article Snippet: ● Bioanalyzer 2100 instrument (Agilent, cat. no. G2939BA) ● Bioanalyzer DNA 1000 Kit (Agilent, cat. no. 5067-1504) ● Bioanalyzer High Sensitivity DNA Kit (Agilent, cat. no. 5067-4627) ● Centrifuge, refrigerated, with fixed-angle rotor (Eppendorf, model no. 5427R) ● Centrifuge with fixed-angle rotor (Eppendorf, model no. 5425) ● Centrifuge with swinging bucket (Eppendorf, model no. 5810R) ● Heater block with wells for 1.5 ml tubes (e.g., Thermo), set to 37 and 56 °C ● Magnetic rack for PCR tubes ● Magnetic rack for 1.5 ml tubes; Dynamag (Thermo, cat. no. 12321D) ● Microcentrifuge (e.g., Roth) ● 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 22-282) ● 1.5 ml microcentrifuge safe-lock tubes (Eppendorf, cat. no. 30120086) ● 1.5 ml microcentrifuge DNA LoBind tubes (Eppendorf, cat. no. 30108051) ● 0.2 ml PCR tubes (Eppendorf, cat. no. 30124359) ● Thermal cycler (Biorad, C1000 touch, cat. no. 1851148/1851196) ● Vortex mixer (e.g., Vortex Genie; VWR) ● Water baths (e.g., VWR), set to 37 °C 5684 NATURE PROTOCOLS | VOL 16 |DECEMBER 2021 | 5673–5706 |www.nature.com/nprot

Techniques: Amplification, Agarose Gel Electrophoresis, Sequencing