STAP-seq input library was generated by cloning the amplified synthetic oligo pool into a human STAP-seq screening vector (Addgene ID 125150) as described previously34 (link),51 (link). 80 μg of input library was transfected into 4x107 BRD4-AID-tagged HCT116 cells using MaxCyte STX. Two independent transfections (biological replicates) were performed. After 30 min recovery phase cells were split in 2 conditions, receiving medium containing water or IAA (500 μM final conc.). Total RNA was isolated 6 h post electroporation followed by polyA+ RNA purification and turbo DNase treatment (Ambion; AM2238). Spike-in control was added in a 1:100 ratio to the isolated total RNA. STAP-seq RNA processing and cDNA amplification was performed as described previously51 (link). Samples were sequenced paired-end on an Illumina NextSeq 550 platform following manufacturer’s protocol and base-calling was performed with CASAVA 1.9.1.
Protocol detail
BRD4-AID-tagged HCT116 cell STAP-seq
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Biological
Brd4
Cdna
Cells
Dnase
Electroporation
Hct116 cells
Human
Library
Oligo
Polya rna
Transfections
Vector
Corresponding Organization : Medical University of Vienna
Other organizations : Research Institute of Molecular Pathology, Whitehead Institute for Biomedical Research
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Variable analysis
independent variables
- IAA (500 μM final conc.)
- STAP-seq RNA processing and cDNA amplification
- Gene expression
- Spike-in control (1:100 ratio to the isolated total RNA)
- BRD4-AID-tagged HCT116 cells
- STAP-seq screening vector (Addgene ID 125150)
- Water treatment condition
- Not explicitly mentioned
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