title: Illumina HiSeq 4000 paired end sequencing; RNA targets of Gal-3 in differentiated mouse embryonic stem cells (mESC)
design: RNA targets of Gal-3 in differentiated mouse embryonic stem cells (mESC)
library_construction_protocol: mESC-R1 cells (kindly provided by Alexander Aulehla, EMBL) were maintained at 37 C and 5% CO2 without feeder cells in high glucose DMEM (Gibco, 41965) supplemented with 15% ES Cell-qualified EmbryoMax Fetal Bovine Serum (Merck Millipore, ES-009-B), penicillin/streptomycin (Sigma Aldrich, P0781), 2 mM L-glutamine (Sigma-Aldrich, G7513), non-essential amino acids (Gibco, 11140), 1 mM sodium pyruvate (Gibco, 11360), 0.1 mM 2-mercaptoethanol and LIF (EMBL Protein Expression and Purification Core Facility), on culture dishes (Nunc) coated with 0.1% gelatin (Sigma-Aldrich, G1890) solution. mESCs were seeded at a concentration of 10.4 cells/mm2 on 15cm dishes in medium without LIF. Cells were incubated for 7 days in the absence of LIF, exchanging the medium every other day, and processed. mESCs were washed twice with ice-cold PBS, place on ice, irradiated (or not) with 150 mJ/cm2 254 nM UV light and lysed on the plate with Dyna lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 1% NP40, 0.1% SDS, 0.5% Na-deoxycholate) supplemented with EDTA-free protease inhibitor (Roche). Lysates were sonicated in a Branson sonifier, cleared by centrifugation, adjusted to a protein concentration of 2 mg/ml, snap frozen in liquid nitrogen and stored at -80 C until use. 1 ml of lysate with 2 mg of total protein was employed per sample. SureBeads Protein G magnetic beads ( Bio-Rad, 161-4023) were washed three times with PBS, 0.1% Tween 20. Lysates were thawed at 37 C and precleared with 25 microliters of washed beads for 30-60 min at 4 C with gentle rotation. 100 microliters of washed beads were coupled to 10 micrograms of anti-Gal-3 (Biolegend, 125402) or Rat control antibody (Biolegend, 400502) in PBS, 0.1% Tween 20 rotating for 1 hour at room temperature, and free antibody was removed by washing the beads three times with Dyna lysis buffer. Precleared lysates were treated with 33.5 U (6.7 microliters) of DNase I (Takara, 2270B) and 0.25 or 0.1 nanograms/microliter of RNase A (Sigma-Aldrich, R5503) for 15 min at 37 C and 1100 rpm. Samples were immediately placed on ice and incubated for 5 min, and then combined with the corresponding coupled beads and incubated for 2 hours at 4 C with gentle rotation. Beads were washed three times with Dyna lysis buffer and two times with PNK buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.5% NP-40). Samples were subsequently processed as described in the supplementary protocol 1 in Van Nostrand EL et al. (PMID: 27018577).     Standard eCLIP library preparation was performed following the supplementary protocol 1 described in Van Nostrand EL et al. (PMID: 27018577).