title: input_F_Saos2_3
organism: Homo sapiens
description: Protocols: RNA immunoprecipitation was performed in HCT116 cells transiently transfected with pCMVtag2b or pCMVtag2bhWig-1 and in Saos-2 TetON cells without insert or Saos-2 TetON cells stably transfected with either Flag-tagged wt Wig-1 or a Flag-tagged Wig-1 zinc-finger 1 point mutant that cannot bind to RNA. Transfected cells were lysed in RIP buffer B (150 mM KCl, 25 mM Tris-HCl, pH 7.5, 10 uM ZnCl2, 1% NP40, 1% Protease Inhibitor Cocktail, 0.5 mM DTT, 100 U/mL RNaseOUT), cleared by centrifugation at 20,000g 30 min at 4C, and filtered through a 45um filter. Lysates containing 30mg of protein were subjected to IP for 2 h rotating at 4C on 600 uL of anti-FLAG M2 beads, pre-equilibrated three times with RIP buffer A (150 mM KCl, 25 mM Tris-HCl, pH 7.5, 10 uM ZnCl2) and once with RIP buffer B rotating at 4 C for 10 min. Input was removed, and beads were washed twice with buffer B rotating at 4 C for 10 min, collected between washes by centrifugation at 50g for 2 min at 4C. RNA was extracted by two rounds of phenol/chloroform/isoamyl alcohol and one round of chloroform extraction, each round consisting of three times 30s vortexing and 30s incubation at 60C, followed by centrifugation at 20,000g for 5 min. RNA was precipitated by using 1 vol of isopropyl alcohol, 0.1 vol of sodium acetate, and 1uL of glycoblue. RNA libraries for sequencing were prepared using TruSeq RNA Sample Prep (Illumina, CA, USA).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-12-31T17:01:13Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T19:30:14Z
attributes.tag: External Id
attributes.value: SAMEA2707074
attributes.tag: INSDC center name
attributes.value: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University
attributes.tag: INSDC first public
attributes.value: 2015-12-31T17:01:13Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T19:30:14Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2840:input_F_Saos2_3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: Saos-2
attributes.tag: common name
attributes.value: human
attributes.tag: individual
attributes.value: 25
attributes.tag: sample name
attributes.value: E-MTAB-2840:input_F_Saos2_3
attributes.tag: scientific_name
attributes.value: Homo sapiens