title: pabpc1a_IP_Rep2 (CRX1181262; CRA018800)
library_construction_protocol: Tg(hsp70:flag-ybx1-EGFP) and Tg(hsp70:flag-pabpc1a-EGFP) embryos were heat shocked at 42 centigrade for 30 min at 36 hpf. The EGFP+ and EGFP- embryos were collected at 52 hpf as experimental group and control group, respectively. 1000 tail region of zebrafish embryos for each sample were resuspended with lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 0.4 U/ml RNase inhibitor) and fully homogenized. After centrifugation, the supernatant was pre-cleared with Dynabeads protein A (Invitrogen) rotating at 4 centigrade for 1 h. 50 microl pre-cleared lysate was spared for Input. Then the remaining lysate was incubated with Anti-Flag M2 Affinity Gel (Invitrogen) at 4 centigrade for 4 h. Washed with ice-cold NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 0.4 U/ml RNase inhibitor) for eight times and following ice-cold MN buffer (50 mM Tris-HCl PH 7.9 and 5 mM CaCl2). Incubated with MNase (2 U/ml, NEBuilder) in MN buffer for 10 min at 37 centigrade, 1100 rpm, the beads were further washed with ice-cold PK buffer (100 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA, 0.2% SDS), and treated with proteinase K (4 mg/ml, Roche) in PK buffer for 1 h at 55 centigrade, 1100 rpm. RNA was extracted using Acid Phenol:ChCl3 (pH 4.3-4.7) and ethanol precipitation. The extracted RNA was subjected to RNA-seq library construction.