title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of human iPSCs derived neural crest stem cells (NCSCs) upon knock-down of BAZ1B (WSTF), including 3 williams beuren syndrome lines, 1 atypical deletion line, and 3 individuals with 7q11.23 duplication syndrome. Each line has been infected with a scramble, or at least one of two short-hairpins (as described in the coupled paper).
design: RNA-seq of human iPSCs derived neural crest stem cells (NCSCs) upon knock-down of BAZ1B (WSTF), including 3 williams beuren syndrome lines, 1 atypical deletion line, and 3 individuals with 7q11.23 duplication syndrome. Each line has been infected with a scramble, or at least one of two short-hairpins (as described in the coupled paper).
library_name: CTL3.sh2_p
library_construction_protocol: Differentiation into NCSCs was performed as previously described (67), with the exception of NCSCs used in the experiment reported in Fig. 1D-F (16).  WBS1, WBS2, WBS3, WBS4, 7dupASD1, AtWBS1 and CTL2 fibroblasts were reprogrammed using the mRNA Reprogramming kit (Stemgent), while the 7dupASD2 and CTL1R lines were reprogrammed with the microRNA Booster kit (Stemgent). The CTL3 line was reprogrammed by transfection with the STEMCCA polycistronic lentiviral vector followed by Cre-mediated excision of the integrated polycistron. 7dupASD3 and CTL4R fibroblasts were reprogrammed using the SimpliconTM RNA Reprogramming kit (Millipore). Prior to differentiation, iPSC lines were cultured on hESC-qualified Matrigel (BD Biosciences) coated plates, diluted 1:40 in DMEM/F-12 and grown in mTeSR-1 medium (StemCell Technologies). They were passaged upon treatment with Accutase (Sigma) and then plated in mTeSR-1 medium supplemented with 5μM Y-27632 (Sigma). Differentiation into NCSCs was performed as previously described (67), with the exception of NCSCs used in the experiment reported in Fig. 1D-F (16).  RNA was extracted using the RNeasy Micro Plus kit (Qiagen) according to manufacturer's instructions. Retrotranscribed cDNA was obtained from 0.5-1 μg of total RNA using the SuperScript VILO Retrotranscription kit (Thermo Fisher Scientific). RT-qPCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems) using SYBR Green Master Mix (Applied Biosystems) as detecting reagent. A total cDNA amount corresponding to 15 ng of starting RNA was used for each reaction. Each sample was analyzed in triplicate and normalized to GAPDH. Relative mRNA quantity was calculated by the comparative cycle threshold (Ct) method using the formula 2-deltaCt. BAZ1B: CCTCGCAGTAAGAAAGCAAAC (forward); ACTCATCCAGCTCCTTTTGAC (reverse). 4 GAPDH: GCACCGTCAAGGCTGAGAAC (forward); AGGGATCTCGCTCCTGGAA (reverse). NR2F1: AGAAGCTCAAGGCGCTACAC (forward); GGGTACTGGCTCCTCACGTA (reverse). NR2F2: GCAAGTGGAGAAGCTCAAGG (forward); GCTTTCCACATGGGCTACAT (reverse). TFAP2A: GCCTCTCGCTCCTCAGCTCC (forward); CGTTGGCAGCTTTACGTCTCCC (reverse). SOX9: AGTACCCGCACTTGCACAAC (forward); GTAATCCGGGTGGTCCTTCT (reverse).  Library preparation for RNA sequencing was performed according to TruSeq Total RNA sample preparation protocol (Illumina), starting from 250 ng - 1 μg of total RNA. cDNA library quality was assessed at an Agilent 2100 Bioanalyzer, using the High sensitivity DNA kit.
attributes.tag: Experimental Factor: disease
attributes.value: normal
attributes.tag: Experimental Factor: RNA interference
attributes.value: BAZ1B knockdown by siRNA 2