Journal: Genome Research

Article Title: Single-cell discovery of m 6 A RNA modifications in the hippocampus

doi: 10.1101/gr.278424.123

Figure Lengend Snippet: Improved m 6 A bulk RNA-seq detection in HEK293T cells. ( A ) Schematic diagram of m 6 A detection with bulk RNA-seq in cultured cells. The YTH protein domain binds to m 6 A. When bound to APOBEC1, the APOBEC1 protein converts C-to-U in the vicinity of m 6 A. This results in a C-to-T mutation in cDNA. C-to-T mutations detected by RNA sequencing are indicative of m 6 A RNA modifications. ( B , left ) Immunofluorescence (IF) of HEK293T cells transfected with Apobec1-Yth-HA-Egfp ( Yth-HA-E ) or Egfp-Apobec1-Yth-HA (E-YTH-HA ). Scale bar, 20 μm. Representative images are shown. ( Right ) Quantification of EGFP and HA overlap. (PCC) Pearson correlation coefficient. ( C ) Number of C-to-T editing events identified in each bulk RNA-seq HEK293T cell replicate for Yth-E and E-Yth plasmids. Editing events identified in at least two replicates were considered for downstream analyses. The data were obtained following Apobec1-Yth-Egfp or Egfp-Apobec1-Yth transfection and EGFP FACS sorting. n = 3. (Rep) separately cultured replicate. ( D ) Metagene analysis showing m 6 A site counts along transcripts for Yth-E and E-Yth bulk RNA-seq results. Nine percent of all m 6 A sites occur in the first 10% of the 3′ UTR following the TTS for YTH-E and 11% for E-YTH, respectively. Shown percentage indicates number of m 6 A sites upstream of, within, and downstream from coding sequence (CDS). ( E ) Metagene analysis showing m 6 A density 500 nt 5′ and 500 nt 3′ from stop codon (0 nt) for YTH-E and E-YTH.

Article Snippet: An Adeno-associated plasmid pAAV- Cag-Egfp-Wpre-Sv40 (gift from Minmin Luo) was used as the backbone to generate the viral expression constructs pAAV- Cag-Apobec1-Yth-Egfp ( Yth-E , Addgene 209322), pAAV- Cag - Apobec1 - Yth mut - Egfp ( Yth mut -E , Addgene 209323), pAAV- Cag-Apobec1 - Egfp (Addgene 209324), pAAV- Cag - Egfp - Apobec1 - Yth ( E-Yth , Addgene 209319), pAAV- Cag-Egfp-Apobec1 - Yth mut ( E - Yth mut , Addgene 209320), and pAAV -Cag-Egfp-Apobec1 (Addgene 209321).

Techniques: RNA Sequencing, Cell Culture, Mutagenesis, Immunofluorescence, Transfection, Sequencing

Journal: Genome Research

Article Title: Single-cell discovery of m 6 A RNA modifications in the hippocampus

doi: 10.1101/gr.278424.123

Figure Lengend Snippet: Detection of m 6 A with bulk RNA-seq in mouse hippocampus. ( A ) Schematic diagram of bulk RNA-seq in the mouse brain. The Egfp-Apobec1-Yth is packaged into AAV viruses to infect brain cells. EGFP-positive cells are isolated from the hippocampus and processed for C-to-U edit and m 6 A site identification. ( B ) Confocal image of mouse hippocampus after AAV infection. Representative image of E-YTH is shown. Half-brain image: Scale bar, 1 mm. Hippocampus image: Scale bar, 400 μm. ( C ) Number of overlapping C-to-U editing events identified by RNA-seq in hippocampus following Egfp-Apobec1-Yth and Egfp-Apobec1-Yth mut AAV virus injection and EGFP FACS sorting. Editing events identified in at least two replicates were considered for downstream analyses. n = 3, (Rep) Biological replicates from different animals. ( D ) Pie chart showing m 6 A localization identified by E-Yth in mouse hippocampus. (TTS) Transcription termination site. ( E ) Metagene analysis showing C-to-U edit scaled density 500 nt 5′ and 500 nt 3′ from stop codon (0 nt) in E-YTH, E-YTH mut , E-APOBEC1, and wild-type (WT) samples. The peak value for E-YTH is 2716 editing events. ( F ) Metagene analysis showing m 6 A density 500 nt 5′ and 500 nt 3′ from stop codon (0 nt). m 6 A sites were obtain after eliminating background from E-YTH editing sites. m 6 A peak density occurs 118 nt downstream from the stop codon. ( G ) Histogram of m 6 A site counts over mutation per read (m/k) ratio. Minimum threshold: 5%.

Article Snippet: An Adeno-associated plasmid pAAV- Cag-Egfp-Wpre-Sv40 (gift from Minmin Luo) was used as the backbone to generate the viral expression constructs pAAV- Cag-Apobec1-Yth-Egfp ( Yth-E , Addgene 209322), pAAV- Cag - Apobec1 - Yth mut - Egfp ( Yth mut -E , Addgene 209323), pAAV- Cag-Apobec1 - Egfp (Addgene 209324), pAAV- Cag - Egfp - Apobec1 - Yth ( E-Yth , Addgene 209319), pAAV- Cag-Egfp-Apobec1 - Yth mut ( E - Yth mut , Addgene 209320), and pAAV -Cag-Egfp-Apobec1 (Addgene 209321).

Techniques: RNA Sequencing, Isolation, Infection, Virus, Injection, Mutagenesis