GSM7060144: BAZ2A_iCLIP_mESC_rep1_background; Mus musculus; OTHER
About 2.5x10^6 mouse ESCs were subjected to 400 mJ/cm2 UV-C (254 nm) crosslinking energy. Cells were subsequently scraped on ice and pelleted at 500 g. To control for unspecific RNA binding 2.5x10^6 mouse ESCs were also subjected to iCLIP analysis without being subjected to UV-crosslinking. The entire procedure was performed in triplicates for each condition. To isolate nuclei cells were resuspended in hypotonic buffer (10 mM HEPES pH 7.6, 1.5 mM MgC2, 10 mM KCl), incubated 10 min on ice, then TritonX-100 was added to the final concentration of 0.1%. Upon additional 10 min of incubation on ice, nuclei were pelleted by 5 min centrifugation at 1600 g at 4°C. Nuclei were lysed into lysis buffer (50 mM Tris pH 7.4, 100 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% NaDeoxycholate) and treated with TURBO™ DNase and RNase I (ThermoFisher, AM2238 and EN0601) (high RNase condition 1:50; low RNase condition 1:400) for 3 min at 37°C at 1100 rpm. To properly solubilize BAZ2A, lysate was subjected to high salt extraction by adding NaCl to final concentration of 400 mM. Upon 10 min incubation on ice, lysate was then centrifuged 10 min at 21000 g at 4°C. Salt concentration was diluted back to physiological 150 mM NaCl by adding iCLIP lysis buffer lacking NaCl. Immunoprecipitation was performed by incubating lysate with the Anti-FLAG® M2 Affinity Gel agarose beads overnight at 4°C under rotation. Beads were washed subsequently twice in High-salt wash (50 mM TRIS PH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% NaDeoxycholate), twice in 0.8 M Urea wash (0.8 M Urea, 20 mM Tris pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% NaDeoxycholate), and twice in PNK buffer (20 mM Tris pH 7.4, 10 mM MgCl2, 0.2% Tween-20). All washes were performed by 5 min incubation at 4°C under rotation followed by 3 min centrifugation at 1300 g at 4°C. RNA 3' end dephosphorylation and L3 adapter ligation steps were performed on beads according to original protocol. 90% of the beads were subjected to RNA 5' end labeling in a total volume of 20 µl of hot PNK mix 2 µl 10× PNK buffer, 16.5 µl H2O, 1 µl PNK, 0.5 µl of 10µCi/µl γ-32P-ATP). Upon 10 min incubation at 37°C at 1100 rpm, beads were quickly washed in PNK buffer. Immunoprecipitated BAZ2A-RNA complexes were eluted from beads by adding a mix containing 20 µl PNK buffer, 6 µl of 4× LDS NuPAGE sample buffer and 1 µl of 1 M DTT and incubated 10 min at 70°C at 1100 rpm. Eluted material was then subjected to SDS-PAGE on a 3-8% TrisAcetate pre-casted gel for 1h at 150 V followed by semidry transfer on nitrocellulose membrane using the iBlot™ Gel Transfer Device (Invitrogen). Membrane was then exposed for 1h or overnight to a Fuji film for autoradiography. Isolation of RNA species associated with BAZ2A from nitrocellulose membrane and reverse transcription were performed according to original protocol. Crosslinked samples were reverse transcribed using the RT#Clip primer #1 (5Phos/NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC), whereas the RT#Clip primer #3 (5Phos/NNATTGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC) was used for non-crosslinked samples. Upon alkaline hydrolysis, precipitated cDNA was subjected to gel purification on a 6% TBE-UREA pre-casted gel according to original protocol. Bands of 80-120 nt were cut out from the gel using the stained DNA ladder and the plastic marks on the gel support for orientation. Gel pieces were then placed into a 0.5 ml microtube, where a hole on its bottom was previously mate using a 21 g needle. Microtube was placed on a 2 ml tube and subjected to centrifugation for 2 min at max speed at room temperature (RT). Crushed gel pieces were then supplemented with 400 µl TE buffer and incubated 30 min at 50°C at 1100 rpm. Eluted material was then passed into a Costar® Spin-X® Centrifuge Tube Filters (Corning, 8161) with two glass pre-filters by spinning 1 min at max speed at RT, and cDNA was subsequently isolated by Phenol:Chloroform extraction following original protocol's instructions. Ligation of the RT#Clip primer to the 5' end of the cDNA via circularization and re-linearization steps were performed according to the original protocol. PCR optimization was performed as indicated and 28 cycles amplification was selected for preparative PCR. Since quality check on gel clearly showed no amplification products for non-crosslinking samples, each crosslinked replicate was pulled in a 1/1 volume ratio with its corresponding non-crosslinked replicate. Libraries were then size-selected and further purified using the BluePippin system (Sage Science) according to manufacturer's instructions. Upon quantification and quality check on 4200 TapeStation System (Agilent), samples were subjected to next generation sequencing.