Unprocessed images for EMM_publication_Proteins are a source of glycans found in preparations of glycoRNA_by_Kegel_et_al

Further information on the manuscript entitled Proteins are a source of glycans found in preparations of glycoRNA Nathanael B. Kegela, Nurseda Yilmaz Demirelb, Timo Glatterb, Katharina Höferb,c,d, Andreas Kaufmanna & Stefan Bauera Affiliations aInstitute of Immunology, Philipps-Universität Marburg, Marburg, Germany bMax-Planck-Institute for Terrestrial Microbiology, Marburg, Germany cCenter for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, Marburg, Germany dDepartment of Pharmacy, Institute of Pharmaceutical Biology and Biotechnology, Philipps-Universität Marburg, Marburg, Germany Summary This file contains unprocessed gel and blots images as well as images with enhanced contrast settings. Additional replicates are shown. Moreover, we provide higher resolution illustrations of the method schematics. Some further method details as well as important observations are explained in the figure legends. Figure 1 Figure 1a HeLa 3T3 Ac4ManNAz 40 h RNA extraction with TRIzol Silica column purification (Zymo) Proteinase K treatment Silica column purification (Zymo) Click chemistry with DBCO-biotin / DBCO-AF647 Silica column purification (Zymo) 45 min, 37 °C 10 min, 55 °C Optional: enzymatic treatment, size fractionation, (…) Gel electrophoresis Northern blot / Fluorescence in-gel detection Further comments on Figure 1a: This schematic illustrates the procedure used for metabolic glycan labelling and RNA extraction. As shown in the paper: Figure 1b 5 kb – 3 kb – 0.25 kb – 1 kb – Northern blot Methylene blue Northern blot Strep 3T3 HeLa nts 3T3 HeLa Glycans Streptavidin-POX, unprocessed Methylene blue, unprocessed Methylene blue, unprocessed, enhanced contrast Methylene blue, unprocessed, overexposure As shown in the paper: Unprocessed images: Further comments on Figure 1b: These unprocessed images show Northern blots obtained from metabolically labeled 3T3 or HeLa cells. In the unprocessed images, the rightmost lanes show unrelated RNA samples that were used as internal controls, which are not relevant for the paper and were therefore not shown in the final figure. In the case of the methylene blue staining, the image with overexposure settings was used in the paper to better visualize the small RNAs. Figure 1c Alexa 647, unprocessed Alexa 647, unprocessed, inverted Ethidium bromide Agarose gel Alexa 647 3 kb – 1 kb – 0.25 kb – >200 3T3 Agarose gel EtBr nts<200 >200 HeLa <200 >200 3T3 <200 >200 HeLa <200 Glycans As shown in the paper: Unprocessed images: Alexa 647, unprocessed, inverted, enhanced contrast Ethidium bromide, inverted Further comments on Figure 1b: These unprocessed images show in-gel fluorescence detections of labelled glycans in RNA extracted from 3T3 or HeLa cells. The samples were run in the lower half of the agarose gel, and the inverted images are shown in the paper. The rightmost lane in these unprocessed images shows a size marker containing a loading dye (bromophenol blue and xylene cyanol), which was not shown in the final figure. These loading dyes emit a fluorescence signal in the Alexa 647 channel. For this reason, such loading dyes must not be used for RNA samples containing AF647-labelled glycans. However, as theses dyes are also visible to the naked eye, they can help assessing migration progress during gel runs. The faint fluorescence signal in samples of large RNAs is most likely unconjugated DBCOAF647. We observed that carry-over of unconjugated fluorescent dye was more common for click reactions in exceedingly large volumes, i.e. >>50 µl. Figure 1d Alexa 647, unprocessed Alexa 647, unprocessed, inverted Alexa 647, unprocessed, inverted, enhanced contrast Alexa 647, unprocessed, inverted, overexposure Ethidium bromide Ethidium bromide, inverted H2O buffer + RNase A/T1 Agarose gel EtBr Agarose gel Alexa 647 –+ Silica column H2O buffer + RNase A/T1 –+ 3 kb – 1 kb – 0.25 kb – As shown in the paper: Unprocessed images: Further comments on Figure 1d: These unprocessed images show in-gel fluorescence detections of labelled glycans in RNA extracted from HeLa cells before and after RNase treatment. The inverted images are shown in the paper. Enhanced contrast settings are shown to visualize faint signals. RNA extraction with TRIzol + silica column 25 µg RNA in 40 µl H2O Add 1 µg proteinase K Add 14 µl H2O or DTB Silica column purification (Zymo) 45 min, 37 °C Click chemistry with DBCO-AF647 10 min, 55 °C Gel electrophoresis Fluorescence in-gel detection Silica column purification (Zymo) Further comments on Figure 2c: This schematic illustrates the procedure used for the new purification strategy using proteinase K treatment under denaturing and reducing conditions via DTB treatment. As shown in the paper: Figure 2c Figure 2d As shown in the paper: Proteinase K –+–+ 0.25 kb – 1 kb – 3 kb – Agarose gel EtBr Agarose gel Alexa 647 H2O DTB 3T3 –+–+ H2O DTB 3T3 Unprocessed images – Replicate #1 Alexa 647, unprocessed Alexa 647, unprocessed, inverted Alexa 647, unprocessed, inverted, enhanced contrast Alexa 647, unprocessed, inverted, overexposure Ethidium bromide Ethidium bromide, inverted Figure 2d As shown in the paper: Proteinase K –+–+ 0.25 kb – 1 kb – 3 kb – Agarose gel EtBr Agarose gel Alexa 647 H2O DTB 3T3 –+–+ H2O DTB 3T3 Unprocessed images – Replicate #2 EtBr Alexa 647 Proteinase K DTB ++ –+ ++ –+ 3 kb – 1 kb – 0.25 kb – Alexa 647, unprocessed Alexa 647, unprocessed, inverted Ethidium bromide Alexa 647, unprocessed, inverted, enhanced contrast Ethidium bromide, inverted Further comments on Figure 2d: These unprocessed images show in-gel fluorescence detections of RNA samples extracted from metabolically labelled 3T3 cells, which were processed according to the new proteinase K + DTB treatment (see Figure 2c). We used the first replicate for the paper, showing the inversed images for better signal visibility. In the second replicate, the strong round signal at ~500 bp is likely unconjugated DBCO-AF647. Figure 3 Figure 3a As shown in the paper: Further comments on Figure 3a: This schematic illustrates the procedure used to rescue labelled glycans after RNase treatment of RNA extracted from metabolically labelled HeLa cells. We observed that labelled glycans were lost during the silica column purification of RNase-treated RNAs (see Figure 1d). As a silica column run essentially consists of the steps Load – Wash – Elute, we argued that the glycans are likely to be found in the column‘s first flow-through after the initial sample loading step. Therefore, we saved the first flow-through (FT) and subjected it to an Amicon® filter column with a 10 kDa molecular weight cut-off. As a control, we also loaded an aliquot of untreated, labelled RNA directly to the Amicon column (see arrow on the right-hand side). As additional controls, we showed the eluate (Elu) from the silica columns, as well as the samples treated with/without RNase treatment before they were loaded to the silica column (Load). Input: purified RNA from HeLa cells, conjugated to DBCO-AF647 RNA Binding Buffer + isopropanol Silica column purification Eluate (Elu) Flow-through (FT) Amicon®Load ±RNase A/T1 Figure 3b As shown in the paper: RNase A/T1 –+–+–+ Input Elu – – Load Load Elu FT FT Input Agarose gel EtBr Agarose gel Alexa 647 0.25 kb – 1 kb – 3 kb – Silica column Amicon® –+–+–+ Input Elu – – Load Load Elu FT FT Input Silica column Amicon® –+–+–+ Input Elu – – Load Load Elu FT FT Input 55 – 100 – 170 – 40 – 35 – Silica column Amicon® SDS-PAGE Alexa 647 kDa Unprocessed images – Replicate #1 (Agarose gel) Alexa 647, unprocessed Alexa 647, unprocessed, inverted Alexa 647, unprocessed, inverted, enhanced contrast Alexa 647, unprocessed, inverted, overexposure Ethidium bromide Ethidium bromide, inverted Figure 3b As shown in the paper: RNase A/T1 –+–+–+ Input Elu – – Load Load Elu FT FT Input Agarose gel EtBr Agarose gel Alexa 647 0.25 kb – 1 kb – 3 kb – Silica column Amicon® –+–+–+ Input Elu – – Load Load Elu FT FT Input Silica column Amicon® –+–+–+ Input Elu – – Load Load Elu FT FT Input 55 – 100 – 170 – 40 – 35 – Silica column Amicon® SDS-PAGE Alexa 647 kDa Unprocessed images – Replicate #1 (SDS-PAGE) Alexa 647, unprocessed Alexa 647, unprocessed, inverted Alexa 647, unprocessed, inverted, enhanced contrast Alexa 647, unprocessed, inverted, overexposure Figure 3b As shown in the paper: RNase A/T1 –+–+–+ Input Elu – – Load Load Elu FT FT Input Agarose gel EtBr Agarose gel Alexa 647 0.25 kb – 1 kb – 3 kb – Silica column Amicon® –+–+–+ Input Elu – – Load Load Elu FT FT Input Silica column Amicon® –+–+–+ Input Elu – – Load Load Elu FT FT Input 55 – 100 – 170 – 40 – 35 – Silica column Amicon® SDS-PAGE Alexa 647 kDa Replicate #2 (Agarose gel) EtBr Alexa 647 Input Elu Elu FT FT Input –++ – –– RNase A/T1 Amicon 10 kDa Input Elu Elu FT FT Input –++ – –– Amicon 10 kDa 0.25 kb – 1 kb – 3 kb – Figure 3b As shown in the paper: RNase A/T1 –+–+–+ Input Elu – – Load Load Elu FT FT Input Agarose gel EtBr Agarose gel Alexa 647 0.25 kb – 1 kb – 3 kb – Silica column Amicon® –+–+–+ Input Elu – – Load Load Elu FT FT Input Silica column Amicon® –+–+–+ Input Elu – – Load Load Elu FT FT Input 55 – 100 – 170 – 40 – 35 – Silica column Amicon® SDS-PAGE Alexa 647 kDa Unprocessed images – Replicate #2 (Agarose gel) Alexa 647, unprocessed Alexa 647, unprocessed, inverted Alexa 647, unprocessed, inverted, enhanced contrast Ethidium bromide Ethidium bromide, inverted Figure 4 As shown in the paper: Figure 5 hLAMP1 130 – 100 – StrepTag 40 – Actin 40 – Actin HeLa sol-hLAMP1 130 – 100 – HeLa sol-hLAMP1 kDa kDa Figure 5a As shown in the paper: Unprocessed images 170 – 130 – 100 – 40 – Anti-hLAMP1 StrepTactin-HRP Anti-Actin Further comments on Figure 5a: This unprocessed Western blot shows three samples of whole cell lysate generated from HeLa cells, and two concentrations of the sol-hLAMP1, which we generated to study glycoprotein co-purification. All samples were loaded twice on the same SDS-PAGE gel and also simultaneously transferred to a PVDF membrane. After blocking, the PVDF membrane was cut along the dashed line, where the left half of the membrane was incubated with an anti-hLAMP1 antibody and the right half was incubated with StrepTactin-HRP to detect the StrepTag. The lower half of the membrane was incubated with an anti-Actin antibody, which served as a loading control. In the paper we show the first two lanes (HeLa and sol-hLAMP1 500 ng), given that 500 ng of the sol-hLAMP1 construct already produced sufficient signal intensity. Input TRIzol 1× 2× 3× 1× 2× 3× EtOH Isoprop StrepTag 130 – 100 – 70 – Silica column kDa Vol. alcohol Figure 5b As shown in the paper: Unprocessed images – Replicate #1 StrepTactin-HRP StrepTactin-HRP, overexposure Colorimetric Input TRIzol 1× 2× 3× 1× 2× 3× EtOH Isoprop StrepTag 130 – 100 – 70 – Silica column kDa Vol. alcohol Figure 5b As shown in the paper: Unprocessed images – Replicate #2 Input TRIzol 1x 2x 3x 1x 2x 3x EtOH Isoprop StrepTag sLAMP1 170 – 130 – 100 – 70 – StrepTactin-HRP StrepTactin-HRP, overexposure Colorimetric Further comments on Figure 5b: These unprocessed images show Western blots of the sol-hLAMP1 construct. We investigated its recovery from the aqeuous phase of TRIzol and its binding to silica columns in an alcohol-concentration-dependent fashion. Images from the frist replicate are shown in the paper. Figure 5c, d As shown in the paper: Unprocessed images – Replicate #1 StrepTactin-HRP StrepTactin-HRP, enhanced contrast Colorimetric 130 – 100 – 70 – RNA added (5 µg) RNase A/T1 –+–+–– Input –+–+* – – + + – – +++–+ 1x 2x StrepTag –+–+–– Input –+–+* – – + + – – +++–+ 1x Agarose gel EtBr 2xVol. isopropanol RNA added (5 µg) RNase A/T1 Vol. isopropanol Silica column Silica column 0.25 kb – 1 kb – 3 kb – c d kDa Ethidium bromide Ethidium bromide, inverted Figure 5c, d As shown in the paper: Unprocessed images – Replicate #2 130 – 100 – 70 – RNA added (5 µg) RNase A/T1 –+–+–– Input –+–+* – – + + – – +++–+ 1x 2x StrepTag –+–+–– Input –+–+* – – + + – – +++–+ 1x Agarose gel EtBr 2xVol. isopropanol RNA added (5 µg) RNase A/T1 Vol. isopropanol Silica column Silica column 0.25 kb – 1 kb – 3 kb – c d kDa StrepTactin-HRP StrepTactin-HRP, enhanced contrast Colorimetric Ethidium bromide Ethidium bromide, inverted Further comments on Figure 5c, d: These unprocessed images show Western blots of the sol-hLAMP1 construct and how its recovery from silica columns is enhanced by the presence of RNA and increased alcohol concentrations. Agarose gels with ethidium bromide staining are provided to show when RNA was added to samples, and to demonstrate RNase activity in the respective lanes. Images from the first replicate are shown in the paper. Supplementary Figure 1 Supp.Figure 1c As shown in the paper: Unprocessed images Proteinase K (µg) 1 20 1 20 0.25 kb – 1 kb – 3 kb – 0 0 Agarose gel EtBr Agarose gel Alexa 647 H2O DTB H2O DTB 3T3 1 20 1 20 0 0 H2O DTB H2O DTB 3T3 Alexa 647, unprocessed Alexa 647, unprocessed, inverted Alexa 647, unprocessed, Inverted, enhanced contrast Ethidium bromide Ethidium bromide, inverted Further comments on Supplementary Figure 1c These unprocessed images show in-gel fluorescence detections of 3T3 RNA, subjected to proteinase K treatment in water or DTB. We compared the efficiency of 1 versus 20 µg of proteinase K for the removal of co-purified glycoproteins. Samples without proteinase K were used as controls. The inverted images are shown in the paper. An image with enhanced contrast settings is provided for better visibility of faint signals. Supplementary Figure 2 Supp.Figure 2a, b Agarose gel EtBr 3 kb – 1 kb – 0.25 kb – Agarose gel EtBr 3 kb – 1 kb – 0.25 kb – a Total 3T3 Large Small Total HeLa Large Small b + Prot. K + Prot. K Unprocessed images HeLa, Ethidium bromide HeLa, Ethidium bromide, inverted 3T3, Ethidium bromide 3T3, Ethidium bromide, inverted As shown in the paper: Further comments on Supplementary Figure 2a, b: These unprocessed images show ethidium bromide stainings of agarose gels used to analyse total, large, and small RNA fractions of 3T3 and HeLa cells submitted to our proteomics pipeline. In the case of the gel showing 3T3 RNA, the gel on the right shows additional samples of small RNAs, used as internal controls. These samples were not relevant for the paper and were therefore not shown in the final figure. Supp.Figure 2c-f As shown in the paper: