Journal: Nature Communications
Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
Article Snippet: Each reaction component for C1-RamDA-seq was as follows: Lysis Final Mix (1.12 μL of 10% NP40, 4.05 μL RealTime ready Cell Lysis Buffer, 0.84 μL of 40 U/μL RNasin Plus RNase Inhibitor, 3 μL of 1:5000 ERCC RNA Spikes, 1.35 μL of C1 Loading Reagent, and 16.55 μL of RNase-free water), gDNA Digestion Final Mix (2.5 μL of 5× PrimeScript Buffer, 5 μL of 1 U/μL DNase I Amplification Grade, 1 μL of 20× C1 Loading Reagent, and 11.5 μL of RNase-free water), Priming Final Mix (17.23 μL of 5× PrimeScript Buffer, 5.24 μL of PrimeScript RT Enzyme Mix, 0.7 μL of 30 μM oligo(dT)12, 2.8 μL of 100 μM 1st-NSRs, 1.75 μL of 2 mg/mL T4 gene 32 protein (NEB), 1.13 μL of C1 Loading Reagent, and 1.15 μL of RNase-free water), RT Final Mix (12 μL of 5× PrimeScript Buffer, 3 μL of PrimeScript RT Enzyme Mix, RealTime ready Cell Lysis Buffer, 0.4 μL of 30 μM oligo(dT)12, 1.6 μL of 100 μM 1st-NSRs, 1 μL of 2 mg/ml T4 gene 32 protein (NEB), 3.96 μL of 1 U/μL DNase I Amplification Grade, 2.25 μL of C1 Loading Reagent, and 33.92 μL of RNase-free water), Second-strand Final Mix (6.7 μL of 10× NEB buffer 2, 6.7 μL of 2.5 mM each dNTP Mixture, 5.36 μL of 100 μM 2nd-NSRs, 2.01 μL of 5 U/μL Klenow Fragment (3′ → 5′ exo-), 1.5 μL of C1 Loading Reagent, and 7.73 μL of RNase-free water), and Harvest Reagent (500 μL of Tagment DNA Buffer, 237.5 μL of C1 Harvest Reagent, and 12.5 μL of 20× C1 Loading Reagent).
Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay