title: GSM8701410: Fos,Input; Mus musculus; ChIP-Seq
library_construction_protocol: A Dounce homogenizer was used to dissociate hippocampal tissue suspended in ice-cold hypotonic NEB fixing buffer containing 250 mM sucrose, 25 mM KCl, 5 mM MgCl₂, 20 mM Hepes-KOH (pH 7.8), 0.5% IGEPAL CA-630, 0.2 mM spermine, 0.5 mM spermidine, 1× protease inhibitor, and 1.1% PFA. The homogenate was incubated at room temperature for 15 min followed by 0.125 M glycine for 5 min to stop the fixation and washed in the centrifuge twice with ice-cold NEB buffer without PFA. The pellet of nuclei was suspended in 400 μL of RIPA lysis buffer (1% IGEPAL CA-630, 0.1 SDS, 0.5% sodium deoxycholate; PBS 1x; pH 8) prior to its sonication in a Sonifier digital SFX 250 Branson for 9 cycles of 15s on/30 s off. After 15 min of centrifugation at 17000 x g, the supernatant containing isolated chromatin was collected. Samples were diluted in 4000 μL of ChIP Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl; pH 8) and incubated overnight at 4°C with the α-Fos monoclonal antibody (Thermo scientific T.142.5). The next day the chromatin was incubated 3 hours with 60 μL of slurry Dynabeads coated to Protein G (Invitrogen) at 4ºC. Beads were thoroughly rinsed at 4ºC as follows: two washes in RIPA-150 buffer, three washes in RIPA-500 buffer (50mM Tris-HCl, 500 mM NaCl, 1 mM, EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate; pH 8), two washes in RIPA LiCl buffer (50 mM Tris-HCl, 1 mM EDTA, 1% NP-40, 0.7%, sodium deosycholate, 500 mM LiCl2), and two final washes in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA; pH 8.0), five minutes each. Samples were resuspended in 200 μl Elution buffer (1%SDS, 0.1M NaHCO3) and treated with 1 μl of 10 mg/mL RNase A (Fermentas) overnight at 65°C in agitation for crosslink reversion. Samples were treated with 2 μl of 20 mg/mL Proteinase K (Thermo Scientific) for 5 hours at 55°C in agitation. Last, DNA precipitation was performed by phenol-chloroform-isoamyl alcohol (Sigma-Aldrich). Hiseq v4
identifiers.namespace: GEO
identifiers.id: GSM8701410_r1