title: GSM7847862: E2F7-GFP, GFP antibody, replicate 2, CUT&RUN; Mus musculus; OTHER
library_construction_protocol: CUT&RUN was performed as described (Meers et al., 2019; Peter J. Skene and Henikoff, 2017), with protocol and buffers detailed in Protocol 2: High Ca2+/low salt (https://www.protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-14egnr4ql5dy/v3). Briefly, E2F7-GFP- or NLS-GFP-transduced mTECs were dissociated in 0.05% trypsin-EDTA in Cell Dissociation Buffer for 15 min at 37°C. Cells were collected in mTEC Basic medium   with 2% Nu-Serum, washed twice in 5 mL of CUT&RUN Wash Buffer, and resuspended in CUT&RUN Wash Buffer. One million cells of each sample were aliquoted in 1.5 mL Eppendorf tubes and brought up to a volume of 1 mL with CUT&RUN Wash Buffer. Cells were incubated with 10 L activated wheat germ agglutinin  beads (Bangs Laboratories, Cat #BP530) at room temperature for 10 minutes with gentle rocking. Cells were combined and incubated overnight with anti-GFP (Abcam, Cat #ab290) diluted 1:100 in 0.05% digitonin in CUT&RUN Wash Buffer.  Following two washes in CUT&RUN Wash Buffer, samples were incubated for 1 hour at room temperature in 1:100 rabbit anti-guinea pig IgG in 0.05% digitonin in CUT&RUN Wash Buffer. Samples were incubated with Protein A/G-MNase fusion protein (Addgene, Cat #123461) at 700 ng/mL for 1 hour at 4°C. Following two washes in 0.05% digitonin in CUT&RUN Wash Buffer and one wash in CUT&RUN Low-Salt Rinse Buffer, samples were digested for 10 min at 0°C in cold CUT&RUN Incubation Buffer. DNA fragments were released by incubation at 37°C for 30 min and then collected by phenol-chloroform extraction. Library preparation was performed with the Next Ultra II DNA Library Prep Kit for Illumina (NEB, Cat #E7645S) using 25 L of CUT&RUN DNA as input, as detailed by N. Liu (https://www.protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-kxygxm7pkl8j/v2). Adaptors were diluted 1:15 in adaptor dilution buffer. High sensitivity D5000 ScreenTape reagents (Agilent, Cat #5067-5593) and the Tapestation 4200 (Agilent) were used to assess library quality and pool libraries for sequencing. Sequencing was done using the NextSeq 550 (Illumina). Library preparation was performed with the NEB Next Ultra II DNA Library Prep Kit for Illumina (NEB, Cat #E7645S) using 25 uL of CUT&RUN DNA as input, as detailed by N. Liu (https://www.protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-kxygxm7pkl8j/v2)