Chromatin profiling provides a versatile means to chart gene regulatory elements and investigate their mechanisms of regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. This is a major limitation as transcriptional states and phenotypes vary markedly across individual cells. Here we combined microfluidics, DNA barcoding and sequencing to profile chromatin at single-cell resolution. We demonstrate the technology by profiling a mixture of ES cells, fibroblasts and hematopoietic progenitors, and deconvoluting in silico high-quality maps for each cell type. We find that regulatory elements differ in their variability, with bivalent promoters showing particularly heterogeneous patterns across single-cells that in contrast with to the relative stability of active promoters and enhancers. Finally, we document a spectrum of ES cell states whose chromatin landscapes exhibit varying degrees of pluripotency and priming signatures. Our study presents an innovative single-cell analysis tool for single-cell analysis, and reveals aspects of epigenetic heterogeneity not be captured by transcriptional analysis alone.
Overall design: Mouse embryonic Stem cells (V6.5) mouse embryonic fibroblasts (globalstem) and hematopoietic progenitor cells (EML) dissociated prior to encapsulation using trypsin and re-suspended in PBS at a concentration of about 5•106/mL. The cell suspension is loaded in a syringe together with the magnetic stirrer bar that prevents sedimentation of the cell suspension. The cell suspension is injected into a co-flow drop-maker device with a lysing buffer and MNase. We use OEM syringe pumps (KD Scientific, MA, USA) with typical flow rates of 1.8 mL/hr for the oil and 250 µL/hr for each of the aqueous phases, resulting in a drop diameter of ~50 µm. Drops are collected and incubated off chip at 4ºC for 10 minutes and 37ºC for 15 minutes to complete cell lysis and chromatin shearing. To label cells in drops, cell-bearing drops and barcode-library drops are re-injected into a “three point merger” device. Drops are spaced on chip by oil with 0.2% w/w surfactant and are then electrocoalesced. An additional ligation buffer is pico-injected as the drops are coalesced. The device electrodes are connected to a high voltage amplifier (TREK 2210) which supplies a 100 V sine wave at a frequency of 25 kHz. The flow rates used to inject the drops are chosen to ensure that no more than one barcode drop fuses with a single cell-bearing drop, even at the expense of some drops not fusing with other drops. Typical flow rates fulfilling these requirements are 1 mL/hr for the oil, 100 µL/hr for the cell-bearing drops, 30 µL/hr for the barcode-drops and 170 µL/hr for the ligation buffer. To control the number of cells collected per sample, we measure the filling number λ at the cell encapsulation stage and use a fast camera (HiSpec1, Fastec Imaging,USA) to measure the frequency f of pairs of barcode-drops and cell-bearing drops that fuse at the labeling stage. The time T100 required to collect 100 cells is then calculated as T100= 100/λf. Typically, f=100 Hz and λ=0.1, so that 1000 pairs of fused drops are collected to sample 100 cells, and the collection lasts 10 seconds. To protect the collected drops from evaporating and adsorbing to the walls of the collection vial, they are collected into a vial containing ~50 µL of oil and 1 % w/w surfactant and 30 µL of emulsion of ~70 µm carrier drops containing only buffer. The sample is collected and incubated at room temperture for 1.5 hour to allow ligation of adapters to chromosomal DNA. Barcodes were commercially synthesized (IDT) and delivered in three 384 well-plates at concentrations of 500 µM. To encapsulate the barcodes in drops we design 96 parallel drop-makers on a single microfluidic chip, so that the aqueous inlets of each drop-maker (22 gauge stainless steel capillaries, New England Small Tube) precisely fit one quarter of a 384 well-plate and are immersed in 96 different wells, each containing a unique barcode. Oil with 1% w/w surfactant is distributed to all drop-makers via a common inlet that is connected to a pressurized oil reservoir. The plate and the microfluidic parallel device are placed in a pressure chamber while a common outlet for all 96 barcode drop-makers is located outside the pressure chamber. Upon pressurizing the chamber, each of the 96 barcode solutions is forced through its own drop-maker, thereby forming an emulsion where every drop contains about 1 billion copies of one of the 96 barcodes. We pressurize the oil reservoir to 9 psi and the pressure chamber to 6 psi, producing ~35 µm drops at a rate of about 500 µL/min from 96 wells. After encapsulation the device is washed with water by placing it in a petri dish filled with water and pressurizing the chamber for 2 minutes. Then the device is similarly washed with isopropanol with the exception that the oil inlet is also fed with isopropanol. Finally, the device is dried by placing it in the pressurized chamber with all inlets exposed for several minutes. The process is repeated 12 times in 6 hours until a total of 1152 different barcodes are encapsulated to form the final barcode-library emulsion. A total volume of about 10 mL is produced from 10 µL in each well.
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