GSM4200517
GSM4200517: IP of WT; Mus musculus; RIP-Seq
Briefly, approximately 15 million HC11 cells (WT or Malat1 mutants) were cross-linked with UV (254 nm, 400 mJ/cm2), followed by lysis in 1 mL of iCLIP lysis buffer, digestion with RNase I (ThermoFisher Scientific, AM2295), and then subjected to immunoprecipitation with 8 μg of a high quality TDP-43 antibody (Proteintech, 10782-2-AP) pre-bound to Dynabeads M-280 Sheep Anti-Rabbit IgG (ThermoFisher Scientific, 11204D). After stringent washes (with 1M NaCl), the TDP-43-RNA complexes were subjected to FastAP (ThermoFisher Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L) treatment, and then ligated to a 3' end RNA adaptor, RiL19 (Table S4) using on-bead ligation with T4 RNA ligase 1 (NEB, M0437M). The TDP-43-RNA complexes from multiple cell lines were then resolved on a NuPage Bis-Tris gel (ThermoFisher Scientific), and transferred to Nitrocellulous membrane. The region of 35-110 kDa was cut from the membrane, which contains the smaller isoform of TDP-43 and its RNA interactome of about 220-nt long (~75 kDa larger than TDP-43 itself), and was then subjected to RNA purification using proteinase K (NEB, P8107S) digestion and phenol-chloroform purification. Purified RNAs were reverse-transcribed using AffinityScript (Agilent, 600107). The rand103Tr3 DNA adapter, containing a random-mer of 10 nucleotides, was then ligated to the cDNA fragment using T4 RNA Ligase 1 (NEB, M0437M). The recovered RNAs were end-repaired by FastAP (ThermoFisher Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L), and then first ligated to RiL19 RNA adaptor, and then to rand103Tr3, similar to the preparation of immunoprecipitated RNAs for sequencing libraries. The final cDNA library was amplified with two oligos, with 6- or 8-nt barcodes in the reverse oligo, for example, PCR_F_D501 and PCR_R_D70, using Q5 High-Fidelity 2X Master Mix (NEB, M0492L). PCR cycles were ~14-16 for TDP-43 IP groups and ~8-10 for SMInput groups. The eCLIP libraries were sequenced on a Next-Seq500 platform with a pair-ended 80-nt run mode.
GEO
GSM4200517
GEO Accession
GSM4200517