title: Illumina HiSeq 2500 sequencing; TDP-43 iCLIP in mESC and hESC
design: TDP-43 iCLIP in mESC and hESC
library_construction_protocol: 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
attributes.tag: Experimental Factor: organism
attributes.value: Homo sapiens