title: CUT&Tag profiling of SYCP1 genomic occupancy in MCF7 breast cancer adenocarcinoma cells upon SYCP1 overexpression
design: CUT&Tag profiling of SYCP1 genomic occupancy in MCF7 breast cancer adenocarcinoma cells upon SYCP1 overexpression
library_construction_protocol: MCF7 lines were maintained in DMEM media supplemented with 2 mM l-glutamine (Invitrogen) and 10% (v/v) fetal calf serum (FCS) at 37 °C in 5% CO2. Cell lines were never maintained for more than 30 passages or 2 months of continuous culturing. Cell lines were tested for mycoplasma on a tri-monthly basis. SYCP1 was overexpressed in MCF7 cells using the pCW-SYCP1_3xTy1 vector. The vector was transfected into MCF7 cells, and a stable clone established. Transfections were performed using Lipofectamine2000 reagent (Invitrogen) following the manufacturer's instructions. SYCP1 expression was induced with 5 μg/mL doxycycline. Control sample was treated with mouse IgG antibody; experimental samples were treated with anti-TY1 antibody. 1x105 of cells were resuspended in a wash buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine). Concanavalin-A beads were prepared by washing twice with binding buffer (20 mM HEPES-KOH pH 7.5, 10 mM KCl, 10 mM CaCl2, 1 mM MnCl2). Ten microlitres of Concanavalin-A beads (Bangs Lab, cat no. BP531) were added to each reaction and incubated with rotation at room temperature for 15 minutes. The cell-bead complex was resuspended in 50 μL antibody buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5 mM spermidine, 0.05% (w/v) digitonin, 0.1% (w/v) BSA). One microgram of anti-Ty1 primary antibody (ThermoFisher Scientific, cat no. MA5-23513) was added to each condition. The reactions were incubated at 4°C overnight with gentle rocking. After removing the supernatant containing the primary antibody using a magnet, secondary antibody was added in dig-wash buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% (w/v) digitonin) and incubated at room temperature for 1 hour with rotation. The complex was then washed twice with dig-wash buffer and resuspended in Dig300 wash buffer (20 mM HEPES-KOH pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% (w/v) digitonin). One microlitre of in-house pA-Tn5 Transposase was added and incubated at room temperature with constant rotation for 1 hour. The complex was washed twice with Dig-300 wash buffer and resuspended in 300 μL Dig-300 wash buffer with 10 mM MgCl2 and incubated at 37°C for 1 hour for tagmentation. The reaction was then quenched by adding 10 μL of 0.5 M EDTA, 3 μL of 10% (w/v) SDS, and 2.5 μL of 20 mg/mL proteinase K and incubated at 50°C for 1 hour. DNA was purified with Serapure beads and eluted in 0.1x TE buffer. Libraries were prepared by tagging each sample with a unique pair of indexes provided in NEBNext® Multiplex Oligos for Illumina® (NEB, cat no. E7311AVIAL) following PCR (Table 2.8). The libraries were purified with Serapure beads. The average fragment length was measured by D1000 ScreenTape (Agilent, cat no. 5067–5582) with D1000 reagents (Agilent, cat no. 5067–5583). Next-generation sequencing was carried out by Macrogen, Singapore.