ItChIP-simultaneous indexing and tagmentation-based ChIP-seq CURRENT STATUS: POSTED

Single-cell measurement of chromatin states, including histone modifications and non-histone protein binding, remains challenging. We present a low-cost, efficient ChIP-seq (simultaneous indexing and tagmentation-based ChIP-seq, itChIP), compatible to both low-input and single cells for profiling chromatin states. This single-cell itChIP approach combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation in a single tube, processing samples with tens of single cells in rarity or with thousands of single cells per assay, and the entire procedure can be finished in two days. The sc-itChIP data acquire ~9,000 unique reads per cell, sufficiently capturing the earliest epigenetic priming along cell fate transition and the basis for cell-type specific enhancer usage. Our results demonstrate that itChIP is a generalizable technology for single-cell chromatin profiling of epigenetically heterogeneous cell populations in many biological processes. This step-by-step protocol is related to the publication “Profiling chromatin state by single-cell itChIP-seq” in Nature Cell Biology.


Introduction
Profiling epigenetic states largely relies on measurement of chromatin occupancy of covalently modified histones, histone regulators, and transcription factors (TFs) 1-5 . Among them, chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is the method of choice, allowing the rapid identification of transcriptional regulatory elements genome-wide [6][7][8][9] . Standard ChIP-seq requires a million or more cells. Reducing the required input to one to hundreds of cells would open up an array of new applications. For instance, understanding the transcriptional regulatory networks that control lineage specification in developing embryos would be enabled by using ChIP-seq to probe lineage-specific enhancers from low amounts of cells. To define differentiation trajectories and potentials of rare spatiotemporal progenitor cells, single-cell RNA-seq (scRNA-seq) techniques have become widely used to separate subpopulations from transcriptomically heterogeneous cell populations [10][11][12][13] . However, a robust single-cell ChIP-seq tool remains lacking to understand epigenetic heterogeneity in complex tissue or cell populations at single cell level.
In the past decade, epigenomic studies have seen great strides towards reduced input sample size, including nano-ChIP 14 , UNI-NChIP 15 , STAR ChIP 16 , MOWChIP 17 , CUT&RUN 18 and ChIL-seq 19 , but 3 development of a generalizable, low-cost, and robust method effective on single cells or small cell populations lags behind. Microfluidic systems have been used to profile histone marks by ChIP-seq using as few as 100 cells 17 . A study by Rotem et al. demonstrated the proof-of-concept use of microfluidic systems for single-cell ChIP (Drop-ChIP). However, at ~800 reads per cell, the sparse data yield from Drop-ChIP limits its potential applications 20 .
Through overcoming these limitations, we present simultaneous indexing and tagmentation of opened/relaxed chromatin-based ChIP-seq (itChIP-seq) (Figure 1), which enables genome-wide profiling of histone modifications and non-histone proteins from ultra-low input (as few as 100 cells) and various histone modifications of single-cell samples (

Detailed Experiment Procedure for Low Input ItChIP
The important steps of the procedure for low-input itChIP were optimized as shown in Figure 3.
• Cell preparation and chromatin opening.
2. Open chromatin: incubation the cell at 62°C for 10 min in an Eppendorf ThermoMixer with agitation at 600 rpm.
3. Quench SDS: add 1.2 μl of 10% Triton X-100 to the samples and incubated at 37°C for 60 min in a PCR thermal cycler.
5. Collect the sample by centrifugation at 12,000 g for 3 min at 4°C, get rid of the supernatant and 7 resuspend the nuclear pellet with 20 μl Releasing Buffer (0.8% SDS, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 200 mM NaCl) by pipetting ~30 times to disperse the nuclei. 6. Incubate the nuclear lysis at RT for 30 min, followed by brief sonication at 20% power for 4 cycles (15 s ON and 30 s OFF per cycle; Q800R2, Qsonica) to aid in solubilize chromatin. 7. Dilute the SDS concentration by adding 100 μl ChIP Dilution Buffer to the chromatin mixture before centrifugation at 20,000 for 15 min at 4°C to collect soluble chromatin for immunoprecipitation.
• Antibody-based chromatin immunoprecipitation. 13. Suspend the complex with 100 μl ChIP Elution Buffer, and reverse-crosslinked at 70°C for 3 h on a thermal mixer at 1500 rpm.
14. Add 0.1 mg/ml proteinase K to the DNA mixture and incubate at 55°C for 3 h. 15. Purify the DNA with phenol-chloroform. The purified DNA can be directly used for library preparation.
• Bio-itChIP.   17. After PCR, purify the library with 1 X AMPure XP beads once. Size selection was carried out by first 0.5X AMPure XP beads to remove >1kb fragments, and second 0.5X AMPure XP beads to the supernatant to obtain 200-1000 bp fragments for sequencing. 18. Sequence the libraries with paired-end 150-bp reads on Hiseq X-ten or Novaseq 6000 platform (Illumina).
Follow the procedures descried above: Step 1-3.  (Table 3) following read lengths: 69 bp (Read1) + 43 bp (Index1) + 37 bp (Index2) + 69 bp (Read2) ( Supplementary Fig. 6). The first 8 bp of Index1 correspond to the T7 barcode and the last 8 bp to the i7 barcode. The first 8 bp of Index2 correspond to the i5 barcode and the last 8 bp of Index2 correspond to the T5 barcode. About 10% PhiX and 15% spike-in library in which we replaced universal connector A/B sequences with 21/27 bp N were pooled with single-cell itChIP libraries for sequencing to balance the constant bases within each detection cycle.
• Single-cell Truseq library preparation for sequencing using the Illumina standard workflow.
30. Prepare the Truseq libraries by 2 rounds of PCR amplification (Fig. 4). 34. Purify the library with 1X AMPure XP beads once and size selection was carried out by first 0.5X AMPure XP beads to remove >1 kb fragments, and second 0.5X AMPure XP beads to the supernatant to obtain 200-1000 bp fragments for sequencing.
Problem: Small amount of soluble chromatin with larger fragment size.
Possible reason: Tn5 complex lose its potency.

Solution:
Increase the amount of Tn5 complex for tagmentation or prepare new Tn5 transpose complex.
Problem: ChIP-qPCR exhibits lower signal or higher background.

Possible reason:
The antibody amount is not optimal.
Solution: Optimize the antibody concentration for immunoprecipitation.

Time Taken
Day 1 • Cell preparation and chromatin opening • Chromatin tagmentation.
• Library preparation for sequencing.
Anticipated Results 1. The library exhibits good ChIP-qPCR enrichment before sequence.   Optimization of key steps in the itChIP protocol. a, b. Optimization of the SDS concentration for chromatin opening. 10,000 ESCs after fixation for 7 min were treated with SDS as indicated concentrations at the step of chromatin opening at 62°C for 10 min, quenched with Triton X-100, tagmentated with Tn5 complex, and briefly sonicated to release chromatin followed by antibody based immunoprecipitation and DNA extraction by phenolchloroform for both soluble and insoluable chromatin after reverse-crosslinking for size examination on agarose gel. We picked the condition yielding desired DNA fragments of < 1 kb (a), as those > 1 kb were undesired for sequencing, and higher ChIP-qPCR enrichment (b) for itChIP. 0.3% SDS was selected according to this criterion. P values were calculated by one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. c, d.

15
Optimization of the required amount of Tn5 complex for efficient tagmentation. The same as in (a), except for the indicated Tn5 amount. We picked the condition yielding DNA fragments of < 1 kb (c) and higher ChIP-qPCR enrichment (d) for itChIP. 3 μl of 12.5 μM Tn5 transposase was selected for 10,000 cells according to this criterion. P values were calculated by one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. e, f. Optimization of the temperature for chromatin opening. The same as in (a), except for the indicated chromatin opening conditions, 37°C for 1 h and 62°C for 10 min. We picked the condition yielding DNA fragments of < 1 kb (e) and higher ChIP-qPCR enrichment (f) for itChIP. In this regard, 37°C for 1 h and 62°C for 10 min exhibited no difference. For simplicity, we used 62°C for 10 min to open chromatin in this study for all the itChIP experiments. P values were calculated by two-tailed paired t-test. g, h.
Optimization of tagmentation conditions. The same as in (a), except for the indicated temperature and time for tagmentation. We picked the condition yielding DNA fragments of < 1 kb (g) and higher ChIP-qPCR enrichment (h) for itChIP. In this regard, both 37°C for 1 h and 55°C for 30 min can be used for chromatin tagmentation. For simplicity, we used 37°C for 1 h for chromatin tagmentation in this study for all the itChIP experiments. P values were calculated by two-tailed paired t-test. Optimal conditions were boxed. Dish lines in a, c, e, g indicate the position of 1 kb DNA marker. In b, d, f and h, data represent means ± s.d. from n=3 biological replicates. Arrowhead indicates free adaptors. Overview of the design of mosaic Truseq library preparation for sequencing using Illumina's standard recipe.

Supplementary Files
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