Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) for capturing transcription dynamics and enhancer activity

We describe a rapid and sensitive labeling of single-stranded DNA for sequencing (KAS-seq), based on a kethoxal-guanine reaction. KAS-seq procedure involves N 3 -kethoxal labeling, DNA isolation, biotinylation, fragmentation and enrichment, library preparation and sequencing. The whole process can be finished in a day. KAS-seq enables rapid measurement of the dynamics of transcriptionally-engaged Pol II, transcribing enhancers, Pol I and Pol III activities, and non-canonical DNA structures involving ssDNA simultaneously in situ , by using as few as 1,000 cells or mice tissues. The robust and tissue-friendly nature coupled with low input material requirement make KAS-seq a method that can be broadly applied. This step-by-step protocol is related to the publication “Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ ” in Nature Methods .


Introduction
Transcription and its regulation determine cell fate and physiological functions, with dysfunctions in transcriptional regulation associated with various human diseases 1 . To understand global transcription regulation, genome-wide sequencing approaches have been developed to analyze the occupancy of RNA polymerases (ChIP-seq) 2 , or detect the presence and level of nascent RNA. Nascent RNA analysis is usually based on run-on assays 3,4 , metabolic labeling 5,6 , and Pol II-associated or chromatin-associated RNA enrichment [7][8][9][10][11] . Although powerful, these methods also have limitations.
Run-on-based methods and Pol II-associated RNA enrichment typically require millions of cells as starting materials. Pol II ChIP-seq could not distinguish whether RNA polymerases are simply bound or are actively engaged in transcription 3 . Metabolic labeling may not be able to accurately measure transient and low-abundant RNA species, such as enhancer RNAs (eRNAs), especially when using limited materials with modest sequencing depth. As most RNAs undergo post-transcriptional processing, their levels are indirect readouts that may not accurately reflect transcription dynamics in situ.
Transcriptionally engaged RNA polymerases resolve DNA double helices and generate single-stranded DNA bubbles. Mapping ssDNA throughout the genome provides a readout of the activity and dynamics of transcriptionally engaged RNA polymerases.
Here we describe a rapid and sensitive labeling of single-stranded DNA for sequencing (KAS-seq, Figure 1), based on a kethoxal-guanine reaction 12 . We show that KAS-seq simultaneously measures   the dynamics of transcriptionally-engaged Pol II, transcribing enhancers, Pol I and Pol III activities, and non-canonical DNA structures involving ssDNA in situ, by using as few as 1,000 cells or mice tissues.  Incubate the mixture at 37 ˚C for 1.5 h with shaking at 500 rpm to facilitate the "click" reaction. 14. Sequence libraries on Illumina platforms by using single-end mode, aiming to get 30 million reads per sample DNAs can be stored at -20 ˚C at the noted stop points if needed.

KAS-seq with mouse liver
Labeling and DNA isolation 1. Homogenize mouse liver tissue to a cell suspension in ice-cold PBS by using a dounce homogenizer or a pellet pestle.

Low-input KAS-seq
Labeling and DNA isolation 1. Label 1,000, 5,000, or 10,000 cells according to the protocol for bulk mammalian cell cultures.

Biotinylation and purification
3. Prepare the click reaction mixture as follows.

µL 20 mM DBCO-PEG 4 -biotin (DMSO solution, Sigma 760749)
Incubate the mixture at 37 ˚C for 1.5 h with shaking at 500 rpm to facilitate the "click" reaction. 11. Mix the beads with 50 µL fragmented DNA from step 9. Incubated the mixture at room temperature for 15 min with gentle rotation. After incubation, place the mixture on a magnetic rack to remove the supernatant and wash beads 5 times with 100 µL 1× binding and wash buffer.

PCR amplification and library sequencing
12. Adjust the volume of input to 20 µL by adding 15 µL nuclease-free water to the 5 µL input saved in step 9. Re-suspend the washed beads in 20 µL nuclease-free water.