Supplementary Materials and Methods s33

Supplementary materials and methods

Simultaneous detection of mRNA and protein in single cells using immunofluorescencecombined single molecule RNA FISH.

Jakub Kochan*, Mateusz Wawro*, Aneta Kasza

Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Cracow, Poland

*J.K. and M.W. contributed equally to this work.

Molecular cloning (Preparation of plasmid constructs used in this report)

Human MCPIP1 (NM_025079.2) was cloned from human cDNA mix. The PCR product was cloned into entry vector pTZ57R/T using InsTAclone PCR cloning kit (Thermo Fisher Scientific, Wilmington, DE, USA) and then subcloned into destination vector pcDNA3.1/mycHisA (Invitrogen, Carlsbad, CA, USA) obtainining MycHis-tagged expression construct. Following primers were used: MCPIP1for and MCPIP1rev.

Human IL-6 3’UTR fragment was cloned from human cDNA mix and inserted into pmirGLO vector (Promega Corporation, Madison, WI, USA) obtaining pLuc-IL-6-3’UTR. Following primers were used: IL-6-3’UTRfor and IL-6-3’UTRrev.

IL-6 3’UTR deletion mutant without the conserved element (IL-6-3’UTRΔCE) was obtained with QuikChange II XL site directed mutagenenis kit (Stratagene, La Jolla, CA, USA) with the following primers: IL-6-3’UTRΔCEfor and IL-6-3’UTRΔCErev.

All restriction enzymes, T4 DNA polymerase/ligase and CIP were obtained from New England Biolabs, Ipswich, MA, USA. Primers used in cloning (listed in Supplementary Table S1) were obtained from Genomed, Warszawa, Poland. All constructs were verified by sequencing. For expression plasmids recombinant protein expression was confirmed by Western blotting.

Supplementary Table S1. List of primers used in cloning

Supplementary Table S2. List of pPCR primers

Supplementary Table S3. Data used to generate standard curves and calculating qPCR primers efficiencies

Supplementary Table S4. Probe sequences of custom probe blends