Nextseq high output kit

Cell lysis, tagmentation and droplet library preparation were performed following the SureCell ATAC-Seq Library Prep Kit User Guide (17004620, Bio-Rad). Harvested cells and tagmentation related buffers were chilled on ice. Lysis was performed simultaneously with tagmentation. Washed and pelleted cells were resuspended in Whole Cell Tagmentation Mix containing 0.1% Tween 20, 0.01% Digitonin, 1 x PBS supplemented with 0.1% BSA, ATAC Tagmentation Buffer and ATAC Tagmentation Enzyme. Cells were mixed and agitated at 500 rpm on a ThermoMixer (Eppendorf) for 30 min at 37 °C. Tagmented cells were kept on ice prior to encapsulation. Tagmented cells were loaded onto a ddSEQ Single-Cell Isolator (12004336, Bio-Rad). Single-cell ATAC-seq libraries were prepared using the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) and SureCell ddSEQ Index Kit (12009360, Bio-Rad). Bead barcoding and sample indexing were performed following the standard protocol and the number of amplification cycles was adjusted according to cell input. Libraries were loaded on a NextSeq 550 (Illumina) and sequencing was performed using the NextSeq High Output Kit (150 cycles) and the following read protocol: Read 1 118 cycles, i7 index read 8 cycles, and Read 2 40 cycles. A custom sequencing primer is required for Read 1 (16005986, Bio-Rad).

ChIP assays were performed from 10⁷ mESC per experiment, according to previously described protocol with slight modification (Rada-Iglesias et al., 2011 (link)). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched by glycine at a final concentration of 0.125 M. Chromatin was sonicated to an average size of 0.5–2 kb, using Bioruptor (Diagenode). A total of 5 μg of antibody was added to the sonicated chromatin and incubated overnight at 4°C. Subsequently, 50 μl of protein G Dynal magnetic beads were added to the ChIP reactions and incubated for ~4 hr at 4°C. Magnetic beads were washed and chromatin eluted, followed by reversal of crosslinks and DNA purification. ChIP DNA was dissolved in water. ChIP-seq and input libraries were prepared according to Illumina protocol and sequenced using Illumina Genome Analyzer. Following library preparation, samples were pooled and sequenced on an Illumina NextSeq instrument using 76 base-pair single-end reads on a NextSeq high output kit (Illumina).