title: cont_rep1
organism: Homo sapiens
description: Protocols: HeLaS3 cells were grown in DMEM supplemented with 10%FBS, pen/strep and L-glutamine. HeLaS3 cells were transfected with 10nMol/l siRNA using Interferin reagent (Polyplus) according to manufacturer instructions. AGO2 reduction was confirmed by Western blot analysis. (1) Pellet 2x 106 cells . (2) Add 300uL of Permeabilization buffer (15mM Tris-HCl pH 7.4, 300mM sucrose, 60 mM KCl, 15 mM naCl, 4mM CaCl2, 0.5mM EGTA, 0.2%NP-40, 0.5mM beta-mercaptoethanol) on ice. (3) Add 50U of Mnase. (4) Incubate for 20 min at 37 degrees C (HeLa S3). (5) Stop reaction by adding 300uL of Stop Buffer (50mM Tris-HCl pH 8, 20mM EDTA, 1%SDS), place the tube on ice and wait 2 min. (6) RNase treatment: add 75uG RNase A 1h at 37 degrees C. (7) Proteinase K treatment: add 30uG RNase A 1h at 55 degrees C. (8) DNA was purified by adding 1 volume of phenol:chloroform:isoamyl alchol, vortex and centrifuge max speed 5-10 min 4 degrees C . Repeat. Add 1 volume of chloroform, vortex and centrifuge max speed 5-10 min 4 degrees C .
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-03-03T16:32:09Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:41:30Z
attributes.tag: External Id
attributes.value: SAMEA2177664
attributes.tag: INSDC center name
attributes.value: University of Rome La Sapienza. Dipartimento di Biotecnologie Cellulari ed matologia, Sezione di Genetica Molecolare
attributes.tag: INSDC first public
attributes.value: 2014-03-03T16:32:09Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:41:30Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-1844:cont_rep1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HeLaS3
attributes.tag: common name
attributes.value: human
attributes.tag: sample name
attributes.value: E-MTAB-1844:cont_rep1
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: specimen with known storage state
attributes.value: frozen specimen