Cellular lysates were fractionated as previously described.25 (link) For strand-specific quantitative reverse transcription polymerase chain reaction (qRT-PCR), only the forward primer was used to amplify the antisense strand and only the reverse primer to amplify the sense strand. 5’ and 3’ rapid amplification of cDNA ends (RACE) was done using Invitrogen RACE System kits. GECPAR was cloned into the pGEM-T vector and subcloned in pCDH-CMV-MCS-EF1-copGFP. pCDH empty backbone or pCDH_GECPAR were transfected in HEK293T, and viral supernatant was then used to infect lymphoma cells. RNA sequencing (RNA-Seq) in cell lines was performed using the NEBNext Ultra II Directional RNA Library Prep.
Protocol detail
Strand-Specific Transcriptional Analysis
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Backbone
Cdna
Cell lines
Cells
Library
Lymphoma
Pgem
Primer
Reverse transcription polymerase chain reaction
Vector
Corresponding Organization :
Other organizations : Istituto Oncologico della Svizzera Italiana, SIB Swiss Institute of Bioinformatics, Cornell University, University of Modena and Reggio Emilia, Azienda Ospedaliera Citta' della Salute e della Scienza di Torino
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Variable analysis
independent variables
- Overexpression of GECPAR
- Transfection of pCDH empty backbone or pCDH_GECPAR in HEK293T cells
- Antisense and sense strand expression levels (measured by strand-specific qRT-PCR)
- RNA sequencing in cell lines
- Cellular lysates fractionation protocol (as described in reference 25)
- Invitrogen RACE System kits for 5' and 3' RACE
- PCDH empty backbone transfection in HEK293T cells
- Not explicitly mentioned
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