GSM2766470: Input DNA; Mus musculus; ChIP-Seq
6-week-old male C57BL/6 mice were terminally anesthetized (ketamine/xylazine, 100/15mg, i.p.) and perfused with only cold PBS just prior to removal of the spinal cord  Tissue preparation and ChIP protocol closely adhered to steps outlined in the MAGnify™ Chromatin Immunoprecipitation System manual (Invitrogen, 49-2024) according to manufacturer's instructions with only a few modifications (see below). Briefly, dissected spinal cords were immediately weighed, cleaned, and minced with a razor blade in ice cold PBS.  Minced tissue was then collectively homogenized by passing repeatedly through 18G and 21G needles before crosslinking chromatin with formaldehyde at a final concentration of 1% at room temperature for 10 minutes.  Glycine was added to a final concentration of 0.125?M to quench formaldehyde crosslinking, and cells were then pelleted and washed with ice-cold PBS.  Cell pellets were resuspended in Lysis Buffer plus Protease Inhibitors at a concentration of 1x10^6 cells / 50 µl.  Nuclear lysates were then sonicated using a Branson® 150 Sonifier (power setting 4, 100% duty cycle for 10 × 30-s on 90-s off intervals), yielding chromatin fragments of 50–300 bp. Dynabeads® Protein A/G (Invitrogen, 10010D) were coupled to either 5 µg of Polyclonal Rabbit anti-Olig2 antibody (Millipore, A9610) or Rabbit IgG (Invitrogen, 100005291) by mixing end-over-end for 4 hours at 4°C prior to introduction to lysates.  Antibody-conjugated beads were then combined with 500 µl of lysate (~1x10^7 cells) plus 500 µl of Dilution Buffer in 1.5 mL LoBind Eppendorf tubes (Sigma-Aldrich, Z666548-250EA) and mixed by end-over-end rotation overnight at 4°C. Approximately 1% of the total sheared chromatin was set aside and served as Input Control.  Beads containing Protein–DNA complexes were subsequently washed with IP Buffer, formaldehyde crosslinking reversed by heat treatment, and the DNA eluted using a 12 Tube Magnetic Separation Rack (NEB, S1509S).  Here, we note that the Magnify™ protocol is designed for use with 0.2 uL tubes, so in all subsequent steps involving end-over-end rotation we instead placed each 1.5 mL LoBind tube on its side and applied gentle agitation using a platform rocker.  This small change to the protocol ensured that the beads remained submerged in solution throughout.  For all steps involving bead collection via magnetic rack, 1.5 mL LoBind tubes were placed in the rack in such a way as to concentrate bead collection toward the bottom of the tube, again to keep them submerged in solution.  Specifically, foam padding was added at the base of the rack to keep the tubes slightly elevated. Input Control samples were processed at the reverse crosslinking step in parallel with the ChIP'd samples. IgG IP'd DNA was analyzed by qPCR but was not included in NGS analysis. Illumina TruSeq ChIP Library Preparation Kit (Illumina, IP-202-1024) according to manufacturer's instructions. Briefly, DNA libraries were prepared from total sheared chromatin (Input Control Library) and ChIP-enriched fragments (ChIP'd Library) using Illumina TruSeq ChIP Library Preparation Kit (Illumina, IP-202-1024). For this experiment, we used a modified TruSeq® ChIP Sample Preparation Guide protocol (Illumina, PN 15023092 Rev.B; Oct. 2013), omitting only the gel purification step to maximize total yield of adapter-ligated library fragments.  Input Control and ChIP'd libraries were prepared using recommended inputs of 100 pg/µl (50 µl), for a total of 5 ng per library prep.  Libraries were indexed using TruSeq Adapters AR008 (Input Control Library; ACTTGA/A) and AR001 (ChIP'd Library; ATCACG/A).  All QC steps (Input DNA quantification, size distributions, and library traces) were evaluated using a Fragment Analyzer Automated CE system with High Sensitivity NGS Fragment Analysis Kit 1bp-6000bp (Advanced Analytical, DNF-474).  Libraries were sequenced at 2 x 50 bp read lengths using the Illumina MiniSeq Sequencing System and MiniSeq High-Output Reagent Kits (Illumina, FC-420-1001).