Single cell RNA expression analysis using flow cytometry based on specific probe ligation and rolling circle amplification

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1 Supporting Information Single cell RNA expression analysis using flow cytometry based on specific ligation and rolling circle amplification Yansong Zhao 1,, Chen Lin 1,, Pengcheng Wu 1, Xiaoyuan Chen 1, Yuancun Zhao 2, Yupeng Li 2, Lu Chen 2, Mats Nilsson 3*, Rongqin Ke 1* 1. School of Biomedical Sciences and School of Medicine, Huaqiao University, Quanzhou , China 2. Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Laboratory Medicine, West China Second University Hospital, State Key Laboratory of Biotherapy, Sichuan University, Chengdu , China 3. Science for Life Laboratory, Department of Biophysics and biochemistry, Stockholm University, Sweden, Box 1031, Se Solna, Sweden These authors contributed equally. * Corresponding authors: Rongqin Ke (rke@hqu.edu.cn) or Mats Nilsson (mats.nilsson@scilifelab.se) 1

2 Supplementary Methods Cell culture and stimulation. The cell line K562 was cultured in RPMI 1640 (Thermo Scientific), supplemented with 10% fetal bovine serum (FBS, Thermo Scientific) and 1% Penicillin-Streptomycin solution (Thermo Scientific). For megakaryocyte differentiation, K562 cells were cultured in complete RPMI 1640 with 5 nm phorbol 12-myristate 13-acetate (PMA, Sigma), supplemented with 10% FBS and 1% Penicillin-Streptomycin solution. For erythroid differentiation, K562 cells were cultured in complete RPMI 1640 with 50 μm hemin (Sigma) and supplemented with 10% FBS and 1% penicillin-streptomycin solution. All cells were incubated at 37 C in a 5% CO 2 humidified atmosphere. Fixation and permeabilization. We usually use cells for each analysis before treatment. Cells were resuspended by pipetting, the cell suspension was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 800g for 5 min at RT. Aspirate the supernatant, 0.5 ml of 4% (w/v) paraformaldehyde (PFA) was added to each tube, mixed well and check for the presence of cell aggregates. If visible aggregates are present, resuspend by gentle pipetting until the cell suspension is homogeneous. Incubated at room temperature (RT) for 20 min and followed by three washes with DEPC-PBS-T (DEPC treated 1 PBS supplemented with 0.1% Tween-20). For permeablization, 0.5 ml HCl (0.1 M) was added to each tube and mixed well, incubated at RT for 30 min and followed by three washes with DEPC-PBS-T. Padlock hybridization. A hybridization mixture containing 0.1 μm padlock s (all padlock sequences can be found in Supplementary Table 2) in 6 SSC and 10% formamide was added to the sample and then incubated for 4 h at 37 C with rotation on a sample mixer, and followed by three washes with DEPC-PBS-T to remove the unhybridized padlock s. For microrna, hybridization was carried in in 1 SplintR buffer (NEB) containing 0.1 μm padlock s, as in our previous publication for microrna in situ detection 1. Padlock ligation. A ligation mixture containing 1 SplintR buffer (NEB), 50% glycerol, 1 U/μL SplintR ligase (NEB), 0.8 U/μL RiboLock RNase Inhibitor (Thermo Scientific), 0.2 μg/μl BSA (NEB) in DEOC-H 2 O was added to the sample and then incubated for 1 h at 37 C with rotation on a sample mixer, and followed by three washes with DEPC-PBS-T. RCA primer hybridization. An RCA primer mixture containing 0.2 μm RCA primers (RCA primer sequences can be found in Supplementary Table 2) in 6 SSC and 10% formamide was added to the sample and incubated at RT for 1 h with rotation, then followed by three washes with DEPC-PBS-T. RCA. An RCA mix containing 1 U/μL phi29 polymerase (Thermo Scientific), 1 U/μL RiboLock RNase Inhibitor, 1 phi29 DNA polymerase buffer (Thermo Scientific), 1 mm dntps, 0.2 μg/μl BSA (NEB), 5% glycerol in DEPC-H2O was added to the sample and incubated for overnight at 37 C and followed by three washes with 1 DEPC-PBS-T. Detection hybridization. A detection mix containing 0.1 μm detection s (all detection s are listed in Supplementary Table 2) in 6 SSC and 10% formamide was added to the sample and incubated at RT for 2 h, then followed by three washes with DEPC-PBS-T. Finally, the cells were suspended in DEPC-PBS and was ready to be analyzed. Flow cytometry acquisition and data analysis. Transfer the cell suspension to polystyrene round-bottom tubes and acquire the data in the flow cytometer, all the data was acquired by using BD FACSCalibur Flow Cytometer. And the flow cytometric data was analyzed with flow analysis software Flow Jo. Microscopy image acquisition. Before analyzed by flow cytometer, 10 μl of the cell sample was transfer to a microscope slide, the slide was then mounted with SlowFade Gold Antifade Mountant (Thermo Scientific) containing 0.5 μg/ml DAPI (Sigma). Images were acquired by using a Leica DM6B microscope equipped with DFC9000GT camera using a 40 objective. CircRNA detection and cell-specific expression. RNA-seq data of K562 and EB cells came from Sichuan 2

3 University. Fastq files of each sample were preprocessed by fastp (v0.18.0) 2 with default parameters for quality control based on duplication rates and gene coverage. Then, they were aligned to the human genome (hg38, Ensemble release 93) using STAR 3 (v2.6.1a, parameters: --outsjfilteroverhangmin , -- chimsegmentmin 12, --chimscoremin 2, --chimscoreseparation 10) to detect chimeric reads. The chimeric junction files from STAR were used to detect BSJs by DCC (v0.4.7) with default parameters 4. Then, raw circrna expression matrices generated per sample were combined in R (version 3.5.1). Only circrnas with more than 5 junction reads were considered as high confident and normalized to reads per million mapped reads (RPM). Cell-specific expression was analyzed by Tau method 5,6 and select the top 25 most differentially expressed circrnas among K562 and EB. References: (1) Lin, C.; Jiang, M.; Duan, S.; Qiu, J.; Hong, Y.; Wang, X.; Chen, X.; Ke, R. Visualization of Individual MicroRNA Molecules in Fixed Cells and Tissues Using Target-Primed Padlock Probe Assay. Biochem. Biophys. Res. Commun. 2020, 526 (3), (2) Chen, S.; Zhou, Y.; Chen, Y.; Gu, J. Fastp: An Ultra-Fast All-in-One FASTQ Preprocessor. In Bioinformatics; (3) Dobin, A.; Davis, C. A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T. R. STAR: Ultrafast Universal RNA-Seq Aligner. Bioinformatics (4) Cheng, J.; Metge, F.; Dieterich, C. Specific Identification and Quantification of Circular RNAs from Sequencing Data. Bioinformatics (5) Yanai, I.; Benjamin, H.; Shmoish, M.; Chalifa-Caspi, V.; Shklar, M.; Ophir, R.; Bar-Even, A.; Horn- Saban, S.; Safran, M.; Domany, E.; et al. Genome-Wide Midrange Transcription Profiles Reveal Expression Level Relationships in Human Tissue Specification. Bioinformatics (6) Kryuchkova-Mostacci, N.; Robinson-Rechavi, M. A Benchmark of Gene Expression Tissue-Specificity Metrics. Brief. Bioinform

4 Supplementary Tables Supplementary Table 1. RNA-seq data for mrna targets in cells. Genes (FPKM) CD44 CD61 PAEP ELF2 circlyn (RPM) K MK EB Note: FPKM and RPM values are mean values obtained from RNA-seq replicates: 3 replicates for K562, 4 replicates for MK and 2 replicates for EB. Supplementary Table 2. Oligonucleotide sequences* Oligonucleotide name CD44 CD61 PAEP CircRNA-LYN Padlock -MiR223 Padlock -ELF2 PPIB GAPDH Sequence ACTGTTGATCACTAGCATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTAC ACAGCTCCATTGCC TGATACAACTGACCCAATGATTACTGACTGC GTCTATTTAGTGGAGCCTACGATCTTCTTTAA CAGGCCACACGTGC TCCACCAGGACTCTGGTTCCTTTTACTCGCGC TTGGTATAATCGCTTACGATCTTCTTTACATG ATCTCATCGTCC AGACTTCCCCAAACAGATACTATTTAGTGGA GCCGACCTTAATCGTGTGATCTCACAGACCG TAATAGTCTCCATGCCCATCC ACAAACTGACATTCCTAGTAATCTCGCGCTT GGTATAATCGCTTTCAAAGTGGGGTATTTG TTCAGTTGACACCTCCATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTCC ATGGGTTCAGACTC TGTCTTGGTGCTCTCCATGATTACTGAC TCGCGCTTGGTATAATCGCTTACGATCT TCTTTGTTTATCCCGGCTGTC TCTTCCTCTTGTGCTCATGATTACTGACTCGC GCTTGGTATAATCGCTTACGATCTTCTTTGCA GTGAGGGTCTCTC 4 Note detected by Cy3 )

5 DapB-1 DapB-2 DapB-3 TTCAGATACGCCATGAATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTAT GGATGGCCAAACGC TTGACGCACCAAGCGGATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTCA TCATGACCAGAAGC 5 AACAACCGGTTTCTCAATGATTACTGACTC GCGCTTGGTATAATCGCTTACGATCTTCTTTT CCGTCCAGTTGTCGG DapB-4 DapB-5 HMBS-1 HMBS-2 HMBS-3 HMBS-4 HMBS-5 RCA-primer-1 RCA-primer-2 RCA-primer-3 TTGGCAGAACGAACACATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTGC AGGAAGCTGTTAAA TCCGTCTTCCGGGACTATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTAT TCGCTTGCACAGCG TAGAGGGAGGCGATAGATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTCC AGAGAAGCAGAGAC TCATCTTTGGGCTGTTATGATTACTGACTCGC GCTTGGTATAATCGCTTACGATCTTCTTTCCC ACGCGAATCACTC TTCTCCAGGGCATGTTATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTCA GGTCCACTTCATTC TCAGGGTGCAGGATCTATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTAG CATACATGCATTCC TTCATAGCTGTATGCAATGATTACTGACTCG CGCTTGGTATAATCGCTTACGATCTTCTTTGT ACAGTTGCCCATCC AGCGATTATACCAAGCGCGA GGCTCCACTAAATAGACGCA CGCACACGATTAAGGTCCAG Cy5 labeled 5 Cy5 -TCGCGCTTGGTATAATCGCT detection Cy3 labeled 5 Cy3-TGCGTCTATTTAGTGGAGCC detection *All oligonucleotides were ordered from Sangon (Shanghai, China). Padlock s were phosphorylated before use. A mixture containing 2 μm of padlock s, 1 PNK buffer A (Thermo Scientific), 1 mm ATP (Thermo Scientific), 0.2 U/μl T4 polynucleotide kinase (Thermo Scientific) was incubated at 37 C for 30 min and 65 C for 10 min. The phosphorylated padlock s can be stored at -20 C until used. 5

6 Supplementary Figures Supplementary Figure 1. Flow chart and timeline for the RCA-Flow protocol. 6

7 Supplementary Figure 2. In situ detection of CD44 and PAEP mrna expression in MK cells. Cells were fixed on slides. Blue for DAPI stained nuclei, cyan dots are detected CD44 and PAEP transcripts CD61. Scale bar 50 μm. 7

8 Supplementary Figure 3. (A) Microscopy examination of CD44 expression in K562 after PMA stimulation. Detection of DapB gene in K562 cells was used as a negative control. Blue for DAPI stained nuclei, cyan dots are detected CD44 transcripts. Scale bar, 50 μm. 8

9 Supplementary Figure 4. Typical microscopy images for detection of HMBS, PPIB and GAPDH expression in U937 cells using the cells prepared by RCA-Flow. DapB was used as a negative control gene. (a) Detection using only one padlock. (b) Detection of DapB and HMBS using five padlock s. Increase in signal was observed. Blue for DAPI stained nuclei, cyan dots are detected mrna signal. Scale bar, 50 μm. 9

10 Supplementary Figure 5. Side scatter plot showing detection of mirna-223 expression in K562 with and without PMA stimulation. Negative control was performed on both K562 and MK lineage by detection of DapB. Due to the change of cell physical properties, side scatter plot of negative control for detection of DapB in MK is also different from that of K562, but the intensity of detected signal remained low. 10

11 Supplementary Figure 5. LYN circular RNA splicing form and sequence. Supplementary Figure 6. Sashimi plot shows splice junction reads and genome coordinate of circrna LYN. Figures on anchors are back-splicing junction counts detected by DCC. 11

12 Supplementary Figure 7. Side scatter plot for negative control DapB and positive control ELF2 detection in K562 before and after hemin stimulation. RNA-seq data also showed that the expression levels of ELF2 remain roughly the same in K562 and its erythroid lineage (erythroblast, EB). 12