GSM3541034: K562_H3K4me2_Rep2_20181202_icell8_329_(20181206_new_TAACAACAGCCCACGT); Homo sapiens; OTHER
Single-cell CUT&Tag: Approximately 50,000 exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding.  Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 1 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 1 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:100 dilution of pA-Tn5 adapter complex was prepared in 1 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 25 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). Cells were loaded to a source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat.  #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 350v chip (Takara Bio USA, Cat. # 640019) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure. Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. We typically obtained ~1000 single cells per chip. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. NEBNext High-Fidelity 2X PCR Master Mix (NEB, M0541L) was dispensed twice using the ICELL8 MSND Single Cell / TCR program for the filtered dispense tool at 50 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 2 min at 98 ◦C followed by 15 cycles of 10 sec at 98 ◦C, 30 sec at 60 ◦C, and 5 s at 72 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions.
GEO Accession
GSM3541034