Illumina HiSeq 2500 sequencing; TDP-43 iCLIP in mESC and hESC
TDP-43 iCLIP in mESC and hESC
The iCLIP protocol was performed as described previously (Tollervey, 2011, Nature Neuroscience), with the following modifications. Cells were UV irradiated once with 160 mJ/cm2 in a Stratlinker 2400 at 254 nm. TDP-43 was immunoprecipitated with protein A Dynabeads conjugated to rabbit-anti TDP-43 (Sigma, SAB4200006). The region corresponding to 55–100kDa complexes was excised from the membrane to isolate the RNA, and sequenced using Illumina HiSeq, instrument to generate 50-nt single-end reads, sequencing depth was 15- 20 Mio reads per library  Mouse ESCs were maintained on gelatin-coated plates in serum free N2B27 added to equal ratio of neurobasal medium and DMEM,  supplemented with MEK inhibitor PD0325901 (1 μM), GSK3 inhibitor CHIR99021 (3 μM), 1,000 U/ml LIF (in-house production), 1% Glutamax, 0.1mM 2-mercaptoethanol , 1% nonessential amino acids.  Human ESCs (line H9) were maintained in mTeSR1 medium (05850, STEMCELL Technologies) on Matrigel-coated plates (356234, Corning) prepared by 1:100 dilution, and 5 ml coating of 10 cm plates for 1 h at 37 °C. Colonies were passaged using the gentle cell dissociation reagent (07174, StemCell Technologies).   mouse ESCs and human ESCs were not treated and grown as described above in pluripotency medium. iCLIP
Experimental Factor: organism
Homo sapiens