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Sample GSM3702243 Query DataSets for GSM3702243
Status Public on Dec 19, 2019
Title TALE2020Ref_161110_JC_002_2
Sample type protein
 
Source name Protein expressed by NEB PureExpress IVT kit (NEB product # E6800L)
Organism synthetic construct
Characteristics protein class: TALE
Extracted molecule protein
Extraction protocol Cloning of TALE proteins: TALE vectors were assembled by a combination of REAL assembly and REAL-Fast assembly . The REAL assembly and REAL-Fast plasmid vectors do not include plasmids encoding non-canonical RVDs, so these non-canonical plasmids were cloned by site-directed mutagenesis on the canonical RVD plasmids. Plasmid vectors expressing one-, two-, or four-long TALE repeats (within the pUC57-ΔBsaI backbone) were ligated in a serial, hierarchical progression to assemble full TALE repeat arrays bearing the proper sequence of repeat variable diresidues (RVDs) to target the DNA sequence of interest. Generally, this assembly involved restriction enzyme digest of each N-terminal TALE repeat vector with BsaI and BamHI, followed by digest of its neighboring C-terminal TALE repeat vector with BsaI and BamHI, and finally ligation of these neighboring repeats by T4 DNA ligase. TALEN expression vectors were digested with SacII and BamHI to obtain the DNA-binding domain comprising the ∆152 N-terminal domain, the RVD repeats, and the +63 C-terminal domain. This fragment was ligated into a modified pDONR221 vector (Invitrogen), with SacII and BamHI restriction sites internal to attL recombination sites, to create Gateway-compatible TALE Entry clones. The TALE constructs were then transferred by Gateway recombinational cloning into the pDEST15 expression vector, which adds an N-terminal glutathione S-transferase (GST) tag (Invitrogen), by an LR reaction. All clones were full-length sequence-verified (Supplementary Data).
Custom PBM design: Target sites for each TALE protein were determined using the canonical TALE code (NI: A, HD: C, NN: G, NG: T), and are preceded by the 5’ T to create the full target site. The constant flanking regions were the same as that used in a prior custom PBM design and do not contain binding sites for any of the TALE proteins in this study . Probe set descriptions, including the array design versions on which they are included, are provided in Supplementary Note. The Agilent AMAD ID for this custom array is 084120.
TALE protein expression: Proteins were expressed using the PURExpress In Vitro Transcription and Translation Kit (New England Biolabs). Protein concentrations were determined by anti-GST western blots with a dilution series of recombinant GST (Sigma). Proteins were stored at 4oC until being used in PBM assays. The duration of storage at 4oC between protein expression and PBM experiments was typically one day, but never greater than three days.
PBM experiments: PBM experiments were performed as follows: briefly, custom-designed microarrays were first double-stranded by an on-slide primer extension reaction. In the PBM assay, arrays were blocked with 2% milk in PBS for 1 h, washed with 0.1% Tween-20 in PBS and 0.01% TX-100 in PBS, then incubated with protein mixture (PBS, 2% milk, 0.2 mg ml-1 BSA, and 0.3 ug ml-1 salmon testes DNA) for 1 h. The final concentration of TALE protein in the PBM reactions was 200 nM, unless otherwise indicated (Supplementary Table 1). Arrays were washed with 0.5% Tween-20 in PBS and 0.01% TX-100 in PBS.
Label Alexa488-conjugated anti-GST primary antibody (Invitrogen A-11131)
Label protocol Lastly, the array was incubated for 20 min with an Alexa488-conjugated anti-GST antibody (Invitrogen A-11131), and washed with 0.05% Tween in PBS and PBS.
 
Hybridization protocol NA
Scan protocol PBM arrays were scanned using a GenePix 4400A Microarray Scanner (Molecular Devices), and scan images were analyzed by GenePix Pro (Molecular Devices).
Description Notes from NEB: PURExpress® is a reconstituted protein synthesis system based on the PUREsystem™ (Shimizu et al., 2001) where all necessary components needed for in vitro transcription and translation are purified from E. coli.
Assay for protein-DNA sequence specificity
Data processing PBM data quantification: Raw data files were processed using the same general approach as used for universal PBMs. Briefly, masliner software was used to combine Alexa488 scans at three different laser power levels and to resolve the signal intensity in spots that are saturated at high laser power settings. Cy3 scans were performed at a single laser power level. If a data set had any negative background-subtracted intensity (BSI) values (which can occur if the region surrounding a spot is brighter than the spot itself), a pseudocount was added to all BSI values for that experiment such that all values were then positive. The custom PBM design included ten replicate probes for each sequence. For each experiment and for each set of probes with identical sequences, we calculated the median-adjusted BSI, median absolute deviation (MAD) and the robust standard deviation estimate from the MAD. Any individual replicate probe with a normalized adjusted BSI value more than 3 s.d. away from the median of the replicate probes was omitted from subsequent analysis, to avoid confounding statistical tests or incorrect choice of parameter settings in model fitting. For each TALE protein, we defined a background set of probes that comprises all the probes on the array designed to represent binding sites for other TALE proteins (not the one being assayed in a given experiment). The array median level was then calculated as the median normalized adjusted BSI of all probes in the background set. The standard deviation of the background set SIs was calculated robustly using the asymptotic approximation σ = 1.4826 x MAD. The z-score for each probe was calculated relative to the median and standard deviation of its corresponding background probes. These z-scores represent a linear transformation of the median SIs for each probe, and therefore facilitate interpretation but do not affect the PWM fitting procedure, which performs its own linear scaling adjustments.
 
Submission date Apr 02, 2019
Last update date Dec 19, 2019
Contact name James Anderson
E-mail(s) jamesanderson12358@gmail.com
Organization name Brigham and Women's Hospital
Department Genetics
Lab Bulyk
Street address 77 Avenue Louis Pasteur
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
 
Platform ID GPL26374
Series (1)
GSE129193 Context and number of non‐canonical repeat variable diresidues (RVDs) impede the design of TALE proteins with improved DNA targeting

Data table header descriptions
ID_REF
VALUE normalized

Data table
ID_REF VALUE
dBr_TALE2072_dinucleotide_substitution_99_replicate_9 5955.320947
dBr_TALE2072_dinucleotide_substitution_99_replicate_8 5629.258198
dBr_TALE2072_dinucleotide_substitution_99_replicate_7 6156.347506
dBr_TALE2072_dinucleotide_substitution_99_replicate_6 5759.966316
dBr_TALE2072_dinucleotide_substitution_99_replicate_5 8325.561486
dBr_TALE2072_dinucleotide_substitution_99_replicate_4 4850.459851
dBr_TALE2072_dinucleotide_substitution_99_replicate_3 5589.256807
dBr_TALE2072_dinucleotide_substitution_99_replicate_2 5843.266073
dBr_TALE2072_dinucleotide_substitution_99_replicate_10 5640.812013
dBr_TALE2072_dinucleotide_substitution_99_replicate_1 5899.492958
dBr_TALE2072_dinucleotide_substitution_98_replicate_9 5062.223913
dBr_TALE2072_dinucleotide_substitution_98_replicate_8 6434.252156
dBr_TALE2072_dinucleotide_substitution_98_replicate_7 5913.184056
dBr_TALE2072_dinucleotide_substitution_98_replicate_6 5105.791422
dBr_TALE2072_dinucleotide_substitution_98_replicate_5 6555.931549
dBr_TALE2072_dinucleotide_substitution_98_replicate_4 5853.001357
dBr_TALE2072_dinucleotide_substitution_98_replicate_3 5434.977767
dBr_TALE2072_dinucleotide_substitution_98_replicate_2 5906.171991
dBr_TALE2072_dinucleotide_substitution_98_replicate_10 6295.584808
dBr_TALE2072_dinucleotide_substitution_98_replicate_1 5636.523223

Total number of rows: 62976

Table truncated, full table size 3961 Kbytes.




Supplementary file Size Download File type/resource
GSM3702243_161110_JC_002_2_TALE2020Ref_TALE2037VA_TALE2037VG_TALE2020HG_TALE2020_VG_TALE2037HG_TALE2037Ref_TALE2020VA_Alexa488_lp450pg100_1-8.gpr.gz 3.2 Mb (ftp)(http) GPR
GSM3702243_161110_JC_002_2_TALE2020Ref_TALE2037VA_TALE2037VG_TALE2020HG_TALE2020_VG_TALE2037HG_TALE2037Ref_TALE2020VA_Alexa488_lp500pg100_1-8.gpr.gz 3.4 Mb (ftp)(http) GPR
GSM3702243_161110_JC_002_2_TALE2020Ref_TALE2037VA_TALE2037VG_TALE2020HG_TALE2020_VG_TALE2037HG_TALE2037Ref_TALE2020VA_Alexa488_lp550pg100_1-8.gpr.gz 3.5 Mb (ftp)(http) GPR
Processed data included within Sample table

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