title: GSM2308743: Mafb_HA-Input; Mus musculus; ChIP-Seq
library_construction_protocol: Mouse retinas were isolated, immersed in liquid nitrogen immediately to snap freeze, and stored at -80 °C for later use. We added 400μl of polysome buffer (50 mM Tris, pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1mM DTT, 200 U/ml RNasin, 1 mg/ml heparin, 100 μg/ml cyclohexamide, and 1x protease inhibitor mixture) to each sample and disrupted the tissue in 1.5 ml microcentrifuge tubes using pellet pestles (749540-0000; Kimble Chase). Postmitochondrial supernatant was formed by centrifugation for 10 minutes at 12,000 rpm at 4 °C and 10μl of the supernatant were saved for input. For IP against HA, we added 25μl of anti-HA antibody-conjugated magnetic beads (M180-11; MBL) to the retinal supernatant prior to incubation on an orbital shaker at 4°C overnight. IP beads were washed for four times with high-salt buffer (50 mM Tris, pH 7.5, 300 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1mM DTT, and 100 μg/ml cyclohexamide) and resuspended in 350μl of RLT plus buffer plus β-mercaptoethanol. Total RNAs were extracted using RNeasy Micro kit (Qiagen) according to manufacturer’s instructions. RNA library construction was performed with the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero (Illumina) according to the manufacturer’s instructions. RNA-Seq / (Sample 19/20) ChIP-Seq
attributes.tag: GEO Accession
attributes.value: GSM2308743