title: GSM6368740: ChIP-seq Ngn2 rep3; Mus musculus; ChIP-Seq
library_construction_protocol: The cortex of post-natal mice aged P5/6 was isolated, mechanically dissociated to obtain a single cell suspension and cultured in medium containing 10% FBS, glucose, pen/strep, B27 supplement, epidermal growth factor and fibroblast growth factor. After culturing for 7 days, the astrocyte enriched culture was reseded on to T-25 flasks and infected with a doxycycline-inducible lentiviral construct. The lentiviral constructs were induced by addition of doxycycline (2ug/ml) for 2 consecutive days in medium containing glucose, pen/strep, glutamine and B27 supplement. Approximately 4 million cells are harvested and cross-linked for 10 minutes at room temperature using 1% formaldehyde. The cross-linking reaction is quenched by the addition of 125mM glycine and the cells are incubated during the quencing reaction at room temperature for 5 minutes. The cross-linked cells are lysed and sonicated using a Bioruptor pico sonication device (settings: 20 cycles, 30 seconds ON/30 seconds OFF) to achieve a fragment size distribution ranging from 150-200bp. The fragment size distribution of the sonicated chromatin is quality controlled using an Agilent 2100 bioanalyser. The chromatin immunoprecipitation was performed by incubating the soniicated chromatin with anti-FLAG antibody (Sigma, F1804) over-night at 4 degree celcius. Thereafter, the antibody-protein-DNA complex was incubated with Dynabeads Protein G for 3 hours at 4 degrees. Following 5 wash steps with LiCL buffer and 1 wash step with TE buffer, the DNA-protein complexes were eluted. The eluate was de-crosslinked by incubation over-night at 65 degree celcius. The de-crossliked DNA was treated with RNase (37 degree celcius for 90 minutes) and Proteinase K (55 degree celcius for 120 minutes). The DNA was purified by UltraPure Phenol:Choloroform:Isoamylalcohol (25:24:1, v/v) (ThermoFisher Scientific) following manufacturer's instructions, followed by ethanol precipitation. The resulting DNA pellet was resuspended in low TE and quantified using Qubit dsDNA HS kit (Thermo Fisher Scientific) following manufacturer;s instructions) The ChIP-seq library was prepared using the MicroPlex Library preparation kit v2 from Diagenode following manufacturer's instructions