title: SH-SY5Y_shCtrl_control_replicate1
organism: Homo sapiens
description: Protocols: HeLa cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 micro-g/mL streptomycin at 37 degrees C and 5% CO2.' Lentiviral vectors (pLKO.1) harboring shRNAs targeting human Unkempt were obtained from Sigma (SHCLNG-NM_001080419). To knockdown Unkempt in mouse neurons, we modified the pSilencer1.0 GFP vector (Shen et al., Nature Genetics, 2010) by inserting between the ApaI and EcoRI sites the shRNAs targeting mouse Unkempt mRNA. The targeted sequences (shUnkW: CCCACTCTTCGTGCAGCACAA, shUnk: GGTATCACCTTCGTTACTA) were identified by the Broad Institute RNAi Consortium. SH-SY5Y cells were transfected at 40% confluence using Opti-MEM I Reduced Serum Medium and Lipofectamine 2000 (both Life Technologies) with a pool of siRNA targeting UNK (siGENOME SMART pool, M-022950-01) or a non-targeting siRNA pool (siGENOME Non-Targeting siRNA Pool 1; D-001206-13; both Thermo Scientific Dharmacon) at 25 nM. After 36 hours of transfection, cells were re-transfected with the above siRNA pools and incubated for an additional 24 hours.' Total RNA extracted from triplicates of each control and knockdown cells was DNase-treated and purified using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA quality was assessed with the 2100 Bioanalyzer (Agilent Technologies).' Sequencing library preparation was carried out using TruSeq RNA Sample Preparation Kit v2 (Illumina).'
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-03-09T17:02:21Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T17:08:55Z
attributes.tag: External Id
attributes.value: SAMEA2341072
attributes.tag: INSDC center name
attributes.value: EBML EBI, Wellcome Trust Genome Campus, Hinxton, CB10 1SD
attributes.tag: INSDC first public
attributes.value: 2015-03-09T17:02:21Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T17:08:55Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2277:SH-SY5Y_shCtrl_control_replicate1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: SH-SY5Y
attributes.tag: common name
attributes.value: human
attributes.tag: genotype
attributes.value: mock treated with shCtrl
attributes.tag: sample name
attributes.value: E-MTAB-2277:SH-SY5Y_shCtrl_control_replicate1
attributes.tag: scientific_name
attributes.value: Homo sapiens