NextSeq 500 paired end sequencing; Activin/Nodal-dependent N6-methyladenosine (m6A) deposition in human pluripotent stem cells
Activin/Nodal-dependent N6-methyladenosine (m6A) deposition in human pluripotent stem cells
m6A MeRIP on nuclear-enriched RNA (NeMeRIP) were performed following modifications of previously described methods (Dominissini et al. 2013; Batista et al. 2014). 7.5E07 hESCs were used for each sample, and three biological replicates per condition were generated. Cells were fed with fresh medium for 2h before being washed with PBS, scraped in cell dissociation buffer (CDB, Gibco), and pelleted at 250g for 5'. The cell pellet was incubated in five volumes of isotonic lysis buffer (ILB: 10mM Tris-HCl pH 7.5; 3mM CaCl2; 2mM MgCl2; 0.32M sucrose; 1,000U/ml RNAsin ribonuclease inhibitor, Promega; and 1mM DTT) for 10' to induce cell swelling. Then, Triton X-100 was added to a final concentration of 0.3% and cells were incubated for 6' to lyse the plasma membranes. Nuclei were pelleted at 600g for 5', washed once with ten volumes of ILB.  Feeder- and serum-free hESC (H9 line; WiCell) and hiPSC (A1ATR/R line, Yusa et al., 2011) culture was previously described (Vallier, 2011). Cells were plated on gelatin- and MEF medium-coated plates, and cultured in chemically defined medium (CDM) containing bovine serum albumin (BSA). CDM was supplemented with 10ng/ml Activin-A and 12ng/ml FGF2 (both from Dr Marko Hyvonen, Dept. of Biochemistry, University of Cambridge). Cells were fed with fresh media containing 10ng/ml Activin-A or 10uM SB-431542. Samples were collected after 2h. RNA was extracted from the nuclear pellet using the RNeasy midi kit (QIAGEN) according to manufacturer's instructions. Residual contaminating DNA was digested in solution using the RNAse-free DNase Set from QIAGEN, and RNA was re-purified by sequential acid phenol-chloroform and chloroform extractions followed by ethanol precipitation. At this stage, complete removal of DNA contamination was confirmed by qPCR of the resulting RNA without a retrotranscription step. RNA was then chemically fragmented in 20μl reactions each containing 20μg of RNA in fragmentation buffer (FB: 10mM ZnCl2; 10mM Tris-HCl pH 7.0). Such reactions were incubated at 95°C for 5', followed by inactivation with 50mM EDTA and storage on ice. The fragmented RNA was then cleaned up by ethanol precipitation. In preparation to the MeRIP, 15μg of anti m6A-antibody (Synaptic Systems, catalogue number: 202-003) or equivalent amounts of rabbit non-immune IgG were cross-linked to 0.5mg of magnetic beads by using the Dynabeads Antibody Coupling Kit (ThermoFisher Scientific) according to manufacturer's instructions. Following equilibration of the magnetic beads by washing with 500μl of binding buffer (BB: 50mM Tris-HCl pH 7.5; 150mM NaCl2; 1% NP-40; 1mM EDTA. MeRIP reactions were assembled with 300μg of the fragmented RNA in 3m of BB supplemented with 500U of RNAsin ribonuclease inhibitor. Samples were incubated at 7rpm for 1h at RT. 5μg of fragmented RNA (10% of the amount used for MeRIP) were set aside as pre-MeRIP input control. MeRIP reactions were washed twice with BB, once with low–salt buffer [LSB: 0.25x SSPE (saline-sodium phosphate-EDTA buffer: 150mM NaCl; 10mM NaHPO4-H2O; 10mM Na2-EDTA; @ pH 7.4); 37.5mM NaCl2; 1mM EDTA; 0.05% Tween-20), once with high-salt buffer (HSB: 0.25x SSPE; 137.5mM NaCl2; 1mM EDTA; 0.05% Tween-20), and twice with TE-Tween buffer (TTB: 10mM Tris-HCl pH 7.4; 1mM EDTA; 0.05% Tween-20). Each wash performed by incubating the beads with 500μl of buffer at 7rpm for 3' at RT. Finally, RNA was eluted from the beads by four successive incubations with 75μl of elution buffer (EB: 50mM Tris-HCl pH 7.5; 150mM NaCl2; 20mM DTT; 0.1% SDS; 1mM EDTA) at 42°C. Both the RNA from pooled MeRIP eluates and the pre-MeRIP input were purified and concentrated by sequential acid phenol-chloroform and chloroform extractions followed by ethanol precipitation. 30μg of glycogen were added as carrier during ethanol precipitation. RNA was resuspended in 15μl of ultrapure RNAse-free water. Library preparation was performed using the Standard TruSeq total RNA kit (Illumina) according to manufacturer's instructions with the following exceptions: (1) Ribo-Zero treatment was performed only for pre-MeRIP samples, as ribosomal RNA contamination in m6A MeRIP samples was minimal; (1) since samples were pre-fragmented, the fragmentation step was bypassed and 30ng of RNA for each sample were used directly for library prep; (3) due to the small size of the library, a 2-fold excess of Ampure XP beads was used during all purification steps in order to retain small fragments; (4) due to the presence of contaminating adapter dimers, the library was gel extracted using gel safe stain and a dark reader in order to remove fragments smaller than ~120bp.
Wellcome Trust Sanger Institute
E-MTAB-5229:Sample 10_p
Experimental Factor: SB-431542
compound
Experimental Factor: 10
dose
Experimental Factor: input RNA
immunoprecipitate