GSM7317494: GATA2_Input; Homo sapiens; RNA-Seq
Cells were washed in 1X PBS and cell pellets were lysed in eCLIP lysis buffer [20 mM HEPES-NaOH pH 7.4, 1 mM EDTA, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1x cOmplete EDTA-free protease inhibitor cocktail (Roche 4693132001)]. Samples were sonicated in a Diagenode Bioruptor (30 s ON/OFF) on medium for 5 minutes. RNase I (ThermoFisher AM2294) was added to lysates for a final concentration of 0.4 U/µL and incubated at 37 C at 1200 rpm for 5 min. EDTA was immediately added at a final concentration of 21 mM. Lysates were clarified at 15,000g for 10 minutes at 4˚C and supernatant was transferred to fresh tubes. Protein concentration was measured using Protein Assay Dye Reagent (Bio-Rad 5000006). Dynabeads were washed in eCLIP binding buffer (20 mM HEPES-NaOH pH 7.4, 20 mM EDTA, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). Antibody was added to bead mixture and incubated, rotating at room temperature for 45 min. Antibody-bead mixture was washed in eCLIP binding buffer and mixed with calculated amount of lysate. Tubes were incubated overnight rotating at 4˚C. 2% of lysate-bead mixture was transferred to a new tube to serve as input sample. IP samples were washed with CLIP wash buffer (20 mM HEPES-NaOH pH 7.4, 20 mM EDTA, 5 mM NaCl, 0.2% Tween-20) and IP50 (20 mM Tris pH 7.3RT, 0.2 mM EDTA, 50 mM KCl, 0.05% NP-40). Samples were treated with TURBO DNase (ThermoFisher AM2238) and 0.1 U/µL final concentration of RNase I (in some cases, 1 U/µL final concentration was used for better visualization of bands, e.g. Fig. S2A). IP samples were washed in CLIP wash buffer and FastAP buffer (10 mM Tris-Cl pH 7.5RT, 5 mM MgCl2, 100 mM KCl, 0.02% Triton X-100). IP RNA was dephosphorylated using FastAP phosphatase reaction FastAP Thermosensitive Alkaline Phosphotase (ThermoFisher EF0652), and T4 PNK (NEB M0201S).     IP samples were washed in CLIP wash buffer and 1X RNA Ligase buffer (50 mM Tris-Cl pH 7.5RT, 10 mM MgCl2]. A 3' IR-800 fluorescent adaptor was ligated using T4 RNA Ligase 1 high concentration (NEB M0437M). Samples were washed in eCLIP high-salt wash buffer (50 mM Tris-HCl pH 7.4RT, 1M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) and CLIP wash buffer. IP and input samples were eluted with 4X LDS Sample Buffer (ThermoFisher NP0007), run on an 8% bis-tris gel, and transferred overnight to a nitrocellulose membrane. The transferred membrane was cut ~0–50 kDa above protein size and incubated with Proteinase K (ThermoFisher AM2548) to isolate crosslinked RNA. Remaining steps were performed as per the seCLIP protocol83, with some modifications. RNA was purified and concentrated with phenol:chloroform:IAA (ThermoFisher AM9732) and ethanol precipitation. 3' and 5' adapters were designed to include an IR800 fluorophore and an 8-nt UMI for cDNA ligation, respectively. We did not include 5' deadenylase enzyme in our 5' ligation reactions and we used the AffinityScript RT (Agilent 600107) for crosslinking-induced truncation.