GSM8244578: Adult_CTX_IgG_0.5_Native2hr_CNR; Mus musculus; OTHER
For CUT&TAG, cortices were collected from 10-week-old wild type mice, and dounced 15 times in HB buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH7.8, 1mM DTT, 0.15 mM spermine, 0.5 mM spermidine, Roche protease inhibitor tablets, 10mM Sodium Butyrate).  IGEPAL CA-630 (Sigma) was added to a final concentration of 0.3% and the lysates were dounced 5 more times.  The lysates were filtered through 40µm cell strainers and spun down at 500g for 5 minutes at 4C.  The pellet was resuspended in cold wash buffer 1 (WB1) (20mM HEPES pH 7.5, 150mM NaCl, 0.2% Tween-20, 0.5 mM spermidine, EDTA-free complete protease inhibitor tablet (Roche), 1mg/mL BSA).  20µl of concanavalin A beads pre-washed with binding buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2) were added to each sample and incubated for 10 minutes at room temperature to bind nuclei to beads.  The supernatant was discarded and 50µl antibody buffer (WB1 with 2mM EDTA, 0.1% Triton) (along with respective antibodies) was added to the beads and incubated overnight at 4C.  Next, the supernatant was discarded, and beads were incubated for 1 hour at room temperature with secondary antibodies diluted 100X in antibody buffer (either pig anti-rabbit antibodies (Novus Biologicals NBP172763) or rabbit anti-mouse antibodies (Abcam ab46540)). After the incubation, beads were washed 3 times with WB1.  The beads were then incubated for 1 hour at room temperature with 0.025µM pA-Tn5 adapter complex diluted in 100 µl wash buffer 2 (WB2) (20mM HEPES pH 7.5, 300mM NaCl, 0.2% Tween-20, 0.5 mM spermidine, EDTA-free complete protease inhibitor tablet (Roche), 1mg/mL BSA). The beads were next washed three times with WB2 and then were incubated for 1 hour at 37C with 300µl tagmentation buffer (WB2 with 10mM MgCl2).  After the incubation, 10ul of 0.5M EDTA, 3µl of 10% SDS, and 2.5µl of 20mg/mL proteinase K were added and incubated for 1 hour at 50°C followed by 70°C for 10 minutes.  The DNA was extracted by phenol-chloroform extractions.  The samples were PCR amplified with KAPA HiFi polymerase along with 1X KAPA HiFi Buffer, 0.3 mM dNTPs, 10µM universal primer (AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG) and 10µM index primer (CAAGCAGAAGACGGCATACGAGAT[index]GTCTCGTGGGCTCGGAGATGT) with the following cycles: (1) 72C for 5 minutes (2) 98C for 30 seconds (3) 98C for 10 seconds (4) 63°C for 30 seconds (5) 72°C for 1 minute (6) 72°C for 5 minutes, (7) go to step 3 thirteen times (8) 12°C hold.  After the PCR, 25µl of Ampure XP beads (0.5x volume) were added and incubated for 10 minutes at room temperature. The supernatant was transferred to a new tube, and an additional 65µl of AMPure beads were added and incubated for 10 minutes at room temperature. The beads were washed twice with 80% ethanol and libraries were eluted with Qiagen EB buffer. ChIP Libraries were constructed by using the Ovation Ultralow Library System (Nugen).  All libraries were sequenced using an Illumina NextSeq 500 CUT&RUN and CUT&TAG