RNA-seq and Luciferase Assay Protocol

Total RNA of kidney samples from different groups were prepared for RNA-sequencing as we previously reported 35 (link), 36 (link). Sequencing and analysis were performed by Novogene (Beijing, China). qPCR and Western blots were performed as we previously described 37 (link). Primers and antibodies used are provided in Tables S1 and S2. For reporter assay, promoter region of Il-6 from -2000 to +500 of TSS (transcription start site) was cloned into pGL3-enhancer (Promega, Madison, WI). Five pGL3-Il-6 luciferase reporter plasmids were constructed by inserting different regions of the Il-6 promoter into pGL3-enhancer vector. NRK52E cells were transfected with indicated plasmids, luciferase assays were performed and analyzed as we previously described 38 (link).

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Wang J., Xiong M., Fan Y., Liu C., Wang Q., Yang D., Yuan Y., Huang Y., Wang S., Zhang Y., Niu S., Yue J., Su H., Zhang C., Chen H., Zheng L, & Huang K. (2022). Mecp2 protects kidney from ischemia-reperfusion injury through transcriptional repressing IL-6/STAT3 signaling. Theranostics, 12(8), 3896-3910.

Publication 2022

pGL3-enhancer

Antibodies Assays Cells Kidney Luciferase Pgl3 Plasmids Primers Promega Transcription start site Vector Western blots

Corresponding Organization :

Other organizations : Huazhong University of Science and Technology, Wuhan University, Hubei Cancer Hospital, Union Hospital

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Variable analysis

independent variables

Groups of kidney samples

dependent variables

Gene expression (RNA-sequencing)
MRNA expression (qPCR)
Protein expression (Western blots)
Luciferase reporter activity

control variables

Kidney samples
RNA extraction and sequencing protocol
QPCR and Western blot procedures
Cell line (NRK52E)
Luciferase reporter assay

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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