alias: GSM3531408
title: GSM3531408: 181102_snm3C_F10_AD006_indexed; Mus musculus; OTHER
library_construction_protocol: in situ Hi-C was performed as previously described using the MboI restriction enzyme. in situ 3C experiments were performed based on the previously described in situ Hi-C protocol with minor modifications. Briefly, prior to fixation, adherent cells were trypsinized, counted, and collected by centrifugation, and suspension cells were counted and collected by centrifugation. Cells were resuspended in culture media at a concentration of 1x106 cells per mL of media and fixed in 1% formaldehyde for 10 minutes at room temperature with shaking. For standard species mixture experiments, equal numbers of mouse and human cells were combined into a single tube prior to fixation. For the 1:10 dilution species mixture experiment, cells were resuspended at a concentration of 1x105 cells per mL of media prior to fixation. For the species mixture experiments where samples were mixed after fixation, each cell type was fixed independently as described above and combined at later stages in the protocol. in situ Hi-C samples were digested with the MboI restriction enzyme and processed as described previously. For in situ 3C experiments, samples were digested with DpnII enzyme overnight at 37ºC with gentle mixing. The following day, the sample was incubated at 62ºC for 10 minutes to inactivate the restriction enzyme. The typical biotin fill in step in the Hi-C protocol is omitted. The sample is then ligated for 4 hours at room temperature with T4 DNA ligase in the same manner as in in situ Hi-C experiments. The sample is then stained with Hoechst (0.1ug/uL) for the final 30 minutes of the ligation step. The sample is then passed through a 40 uM nylon cell strainer (Corning 431750) into a FACS tube prior to sorting. As a quality control step, 10% of the sample is taken for conventional library preparation and sequenced using shallow sequencing on a MiSeq. Libraries for bulk and single-cell methylomes were generated using snmC-seq2. A detailed step-by-step bench protocol for snmC-seq2 is provided as Supplement Methods in Luo et al. (2018) 23. Bulk methylome libraries were prepared manually using individual tubes. Single-cell methylome libraries were prepared using a Tecan Evo 100 robotic platform as described in Luo et al. (2018) 23. Sequencing libraries were sequenced using Illumina MiSeq and HiSeq 4000 instruments in PE150 mode.
identifiers.namespace: GEO
identifiers.id: GSM3531408