title: GSM1891866: 4T1 D40A - sample 10 - input; Mus musculus; RIP-Seq
library_construction_protocol: 4T1 cells expressing 8166-M24-658 control vector or FLAG-CNOT7 constructs were grown to ~85% confluence in ten 15cm culture plates. Cells were then rinsed with 15mL ice cold sterile PBS, scraped off, pelleted (~1mL pellet), and snap frozen in liquid nitrogen. Cells were thawed on ice and lysed in 3.5mL lysis buffer (100mM NaCl, 5mM MgCl2, 10mM HEPES (pH 7.3), 0.5% NP40, 200 units RNasin (Promega), Protease inhibitor cocktail (Roche)). The resulting 4mL of cell lysate was spun down twice at 21,000*g for 20 minutes at 4˚C. 40uL of lysate was saved 1% input to confirm immunoprecipitation. 200uL of lysate was saved for 5% RNA-seq input and was purified by TriPure (Roche) RNA extraction. 25uL beads per 1mL lysate of Protein G Dynabeads® were blocked with 1mL 0.5% BSA in PBS at room temperature for 20 minutes then washed twice with 1mL NT2 wash buffer (50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM MgCl2, 0.05% NP40). 24ug antibody was added to each sample and incubated at 4˚C overnight then beads were added to each sample and rotated for 30 minutes at room temperature. Beads were washed four times in 1mL NT2 buffer. Eighty five percent of beads were subjected to RNA extraction with TriPure. Protein from the remaining 15% of beads was eluted with Laemli buffer to confirm immunoprecipitation. Library preparation was performed using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® with NEBNext® multiplexing oligos using manufacturer’s protocol.  RNA sequencing was conducted on the Illumina® HiSeq 2500.
attributes.tag: GEO Accession
attributes.value: GSM1891866