GSM7068179: Control_rep2; Mus musculus; OTHER
The embryoid bodies (EBs) of 9 days were resuspended in the PBS buffer and treated with UV crosslinking (254nm, 400 mJ/cm2) using a Stratalinker. Crosslinked EBs were lysated in 1ml iCLIP lysis buffer (50mM Tris pH7.4, 100mM NaCl, 1% Igepal CA630, 0.1% SDS, 0.5% sodium deoxycholate, RNase inhibitor, and protease inhibitor cocktail). We performed the CLIP-seq following the eCLIP-seq protocol (Van Nostrand et al., 2016) except several modifications as below. The V6.5 mESCs over-expressing V5-tagged Csrp1 and wildtype V6.5 mESCs were differentiated into embryoid bodies as described above separately. The embryoid bodies (EBs) of 9 days were resuspended in the PBS buffer and treated with UV crosslinking (254nm, 400 mJ/cm2) using a Stratalinker. Crosslinked EBs were resuspended in 1ml iCLIP lysis buffer (50mM Tris pH7.4, 100mM NaCl, 1% Igepal CA630, 0.1% SDS, 0.5% sodium deoxycholate, RNase inhibitor, and protease inhibitor cocktail) and then solubilized for 1 minute via sonication with a Covaris S220 instrument using following parameters: PIP 140W, Duty factor 5%, CPB 200. The lysates were limited digested with 0.4 µl RNase I for 5 minutes at 37oC and then centrifuged for 15 minutes with 15,000g. The 100 µl protein G magnetic beads were incubated with 10 µg anti-V5 tag antibody for 30 minutes at 4oC to prepare anti-V5 magnetic beads, and then added to the cell lysates and incubated for 2 hrs at 4oC. Next, the beads were washed as below: two times with 1ml high salt wash buffer (50mM Tris pH7.4, 1M NaCl, 1% Igepal CA630, 0.1% SDS, 0.5% sodium deoxycholate); two times with 1ml wash buffer (20mM Tris pH7.4, 10mM MgCl2, 0.2% Tween-20); two times with 1ml 1x DNase buffer (10mM Tris pH7.4, 2.5mM MgCl2, 0.5mM CaCl2). Beads were resuspended in 100ul 1x DNase buffer supplemented with 4U DNase I and incubated for 20mins at 37oC. Beads were then washed two times with 1ml PK buffer (20mM Tris pH7.4, 50mM NaCl, 1mM EDTA). Beads were resuspended in 260ul PK buffer and supplemented with 40 µl proteinase K (NEB). Beads were incubated for 2 hrs at 37oC and then mixed well with 200 µl phenol and 200µl 1-Bromo-3-chloropropane (BCP), which was then centrifuged at 13000rpm for 15 minutes at 4°C. The supernatant was transferred to a new tube, mixed with 200μl BCP, vortexed for 30 seconds and centrifuged at 13000rpm for 15 minutes at 4°C. The supernatant was transferred to a new tube and supplied with 30μl 3M sodium acetate (NaOAc) pH5.2, and 2μl glycogen (5mg/ml, ThermoFisher). The mixture was then precipitated by adding 2.5x volumes of 100% ethanol and centrifuged at 13000rpm for 15 minutes at 4°C. The pellets were washed with 80% ethanol twice, and then dissolved in 15µl H2O. The resultant RNAs were first ligated with 3′ barcoded (NNNNNNNNNNNNNNN) RNA adaptor and then 5′ RNA adaptor. cDNAs were synthesized with M-MLV reverse transcriptase (Vazyme) and amplified by VAHTS HiFi amplification mix (Vazyme). The PCR products were cleaned using DNA clean beads (Vazyme) and separated on agarose gel. The library was purified using MinElute Gel Extraction Kit (Qiagen) and evaluated using Qsep with S2 Cartridge. The libraries were finally sequenced on NovaSeq 6000 platform (Illumina) by Shenzhen Haplox Biotechnology.