title: GSM4387136: Cut-and-Run-H3K27me3-AB1-brain-mNPC-Ybx1ko-S2-Rep2-5hrs; Mus musculus; OTHER
library_construction_protocol: Ultra-low input native ChIP (ULI-nChIP) was performed as described in Brind'Amour et.al. 2015.  Briefly, mNPCs from Sox2-GFP positive embryos were isolated and sorted as described above.  Using an equal number of cells as input, nuclei were isolated using Nuclear Isolation Buffer  (MilliporeSigma NUC101) and diluted MNase enzyme added to digest DNA. The MNase  reaction was carried out for 7.5 min at 37°C and stopped by addition of EDTA. A 1% Triton/1%  deoxycholate solution was added to lyse nuclei and complete immunoprecipitation buffer added  to allow for ~200 μl lysate/immunoprecipitation. Antibodies were bound to Protein A/G magnetic  beads (ThermoFisher) and after pre-clearing on washed magnetic beads with no antibody,  antibody-bead complexes were added to chromatin O/N at 4°C. After successive washes in low  salt and high salt wash buffers, chromatin was eluted in ChIP elution buffer for 1.5 h at 65°C.  DNA was extracted using phenol-chloroform-isoamyl alcohol extraction and MaXtract phaselock  tubes (QIAGEN) to retain maximal DNA. DNA was further purified using Ampure XP beads  (Agencourt) and libraries constructed through the following processes: 1) End repair with T4  PNK, Klenow DNA polymerase, and T4 DNA polymerase; 2) A-tailing with dATP and Klenow  (3'-5' exo-) polymerase; 3) Adapter ligation with annealed Illumina adapters and Quick DNA  ligase; and 4) Library amplification with Illumina paired-end indexed primers and 2X Phusion HF  Master Mix. Library amplification was performed for 12 cycles and DNA purified by Ampure XP  beads and library yield and quality evaluated using a D1000 High Sensitivity screentape on an  Agilent TapeStation. CUT&RUN was performed as described in Skene and Henikoff (2018), with minor variations.  Briefly, mNPCs from Sox2-eGFP–positive embryos were isolated and sorted as described  above. Cells were pelleted and resuspended in wash buffer. Bio-Mag Plus Concanavalin-A  coated beads (Bangs Laboratories BP531) were added to cells (diluted in binding buffer) to bind  nuclei to beads. Supernatant was removed and samples were blocked for 5 min at RT with  digitonin block buffer. Antibody diluted in digitonin block buffer was added and samples were  rotated for 3 h, 5 h, or O/N in the cold. Beads were collected and washed 3× with digitonin block  buffer before adding pA-MNase to beads. After a 1-h incubation, beads were washed 3× and  resuspended in wash buffer. After equilibration on ice, 100 mM CaCl2 was added to tubes and  samples were incubated for 25 min with agitation at the 15-min mark. The reaction was stopped  by adding the stop buffer. Samples were incubated at 37°C for 30 min to release chromatin.  DNA was isolated by phenol–chloroform–isoamyl alcohol extraction and MaXtract phase-lock  tubes (QIAGEN) to maximally retain chromatin. Chromatin was resuspended in low EDTA TE  buffer and analyzed by TapeStation using the HS DNA Kit (Agilent). Libraries were made using  the Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences) and submitted for sequencing. In  all cases, ~15% input was removed and IgG was used as a negative control.
attributes.tag: GEO Accession
attributes.value: GSM4387136