title: GSM4010641: mES_am_Ctrl_TBE_Pol2_rep2; Mus musculus; OTHER
library_construction_protocol: Antibody and pA-Tn5 (Vazyme Biotech, S603) or pG-Tn5 (Vazyme Biotech, S602) complex was prepared 20min ahead of sample lysis step. Add 1ul fully dissolved 5% digitonin into 1ml Buffer1 (10 mM Tris-HCl pH=7.4, 150mM NaCl, 0.5mM NaCl, 1x EDTA-free Roche complete protease inhitibor.) to prepare DB-1. Add 7μl DB-1, 0.5ul pA/G-Tn5 (choosing pA or pG depends on antibody species and affinity, we used pG-Tn5 for Pol II and pA-Tn5 for H3K4me3 and H3K27me3), 0.5ug antibody into 200ul low-binding tube and mix by vortex. Incubate at 4°C for 30min. Briefly, for Stacc-seq without wash step, fresh cells or embryos were prepared and put into 200ul low-binding tube (total volume with buffer should be less than 1μl). Add 6ul DB-1 into the sample and vortex. Incubate at 4°C for 10min, vortex each 2.5min for 3 times. Add 35μl DB-1 into the resulting samples, then add pA/G-Tn5 mixture and 12.5μl pre-warmed (37°C) 5x TTBL (Vazyme Biotech, TD502). Incubate in Thermo mixer at 37 °C for 30 min at 400 rpm. DNA was purified by phenol-chloroform with 40ng carrier RNA and 2ul spike-in DNA followed by ethanol precipitation 30min at -80°C or overnight at -20°C. PCR was performed to amplify the library for 16 cycles using the following PCR conditions: 72 °C for 3 min; 98 °C for 30s; and thermocycling at 98°C for 15s, 60°C for 30s and 72°C for 3min; followed by 72 °C 5 min. After the PCR reaction, libraries were purified with the 0.4x-1.7x AMPure (Beckman) beads size selection and were subjected to next generation sequencing. For Stacc-seq with wash step, fresh cells or embryos were suspended in 50ul Buffer1 into 200ul low-binding tube. 10ul Concanavalin-coated magnetic beads (Polyscience, 86057) were washed by 200 ul Buffer2 (10 mM Tris-HCl pH=7.4, 10mM KCl, 1mM CaCl2, 1mM MnCl2) once and resuspended in 10ul Buffer2. Add beads to samples and incubate for 10mins at room temperature. Place the samples on magnet stand and remove the supernatant and then wash beads with 200ul Buffer1 twice. Add pA/G-Tn5 mixture and DB-1 to 50ul to samples and resuspend the beads. Place tube on 4°C Thermo mixer at 400 rpm for 2h. Place the tube on magnet and remove the supernatant. Wash beads twice with 200ul DB-1. Resuspend the beads with 50ul DB-1 and add 12.5ul pre-warmed (37°C) 5x TTBL (Vazyme Biotech, TD502). Incubate in Thermo mixer at 37 °C for 30 min at 400 rpm. DNA was directly purified without removing beads by phenol-chloroform with 40ng carrier RNA and 2ul spike-in DNA followed by ethanol precipitation 30min at -80°C or overnight at -20°C. PCR was performed to amplify the library for 16 cycles using the following PCR conditions: 72 °C for 3 min; 98 °C for 30s; and thermocycling at 98°C for 15s, 60°C for 30s and 72°C for 3min; followed by 72 °C 5 min. After the PCR reaction, libraries were purified with the 0.4x-1.7x AMPure (Beckman) beads size selection and were subjected to next generation sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4010641