Journal: Nature methods

Article Title: Protein-RNA networks revealed through covalent RNA marks

doi: 10.1038/nmeth.3651

Figure Lengend Snippet: The RNA Tagging approach. a) Strategy. RBP, RNA-binding protein. PUP, poly(U) polymerase. b) Schematic of targeted RT-PCR and transcriptome-wide RNA Tagging assays. RNAs are tailed with a combination of guanosines (G) and inosines (I) (purple). The U-select primer contained the Illumina 3′ adapter sequence (brown), nine cytosines (purple) that base pair with the G/I tail, and three adenosines (red) that select for uridines at the 3′ end of the mRNA. c) Computational identification of Tagged RNAs. A-tails refers to the poly(A) tail and U-tails refers to 3′ terminal uridines, which were often in the U-tag. d) Nature of the data. The cartoon depicts Tagged RNAs aligned to a representative gene. ORF, open reading frame. e) Plot of the mean U-tag length detected by high-throughput sequencing of synthetic DNA libraries that contained U-tags of 0, 2, 4, 6, 8, 10, and 12 nucleotides. At least 50,000 reads were detected for each library (>1 million total reads). The R 2 value ( R 2 = 0.99, n = 7) was determined by linear regression analysis, and error bars represent standard deviation.

Article Snippet: 60 μL of cDNA was added to 10 μL of 10X Klenow Buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCL 2 , 10 mM DTT, 0.5 mg per mL BSA), 12 μL of water, 5 μL of 10 mM dNTPs, 10 μL of 10 μM 2 nd strand synthesis primer (GTTCAGAGTTCTACAGTCCGACGATCNNNNNN), and 3 μL of 5 U per μL Exo- Klenow DNA Polymerase (Life Technologies).

Techniques: RNA Binding Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Next-Generation Sequencing, Standard Deviation

Journal: Cell Reports Methods

Article Title: Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

doi: 10.1016/j.crmeth.2023.100465

Figure Lengend Snippet:

Article Snippet: Mouse α-MYH7 monoclonal , Santa Cruz Biotech , Cat# Sc-53090; RRID: AB_2147279.

Techniques: Recombinant, Membrane, Transfection, SYBR Green Assay, DNA Methylation Assay, Mutagenesis, CRISPR, Software, Functional Assay, Microarray, In Vivo, Comparison, Next-Generation Sequencing

Journal: Cell Reports Methods

Article Title: Retrospective cell lineage reconstruction in humans by using short tandem repeats

doi: 10.1016/j.crmeth.2021.100054

Figure Lengend Snippet: Duplex MIPs-based cell lineage workflow (A) Design of duplex MIPs precursor: desired targets are selected from our cell lineage database and precursors are designed. (B) Duplex MIPs preparation: duplex MIPs precursors are synthesized on a microarray, pooled and amplified by PCR. The product is digested by MlyI to remove the universal adaptors (orange and green), purified and diluted to obtain active duplex MIPs. (C) Duplex MIPs and template DNA are mixed together to allow annealing of the targeting arms (blue and yellow) to the regions flanking the targeted sequence in the template DNA, the products are then circularized by gap filling, facilitated by DNA polymerase and ligase. Linear DNA, including excess MIPs and template DNA, is eliminated by exonucleases digestion. The Illumina sequencing library is generated individually for each sample in a PCR step, facilitating addition of adaptors and barcodes. Libraries are pooled and sequenced by using Illumina next-generation sequencing platform, followed by analysis of the raw reads to detect mutations. The latter are then used to infer the cell lineage tree.

Article Snippet: Sample specific barcoding PCR was performed using dual-index barcoding primers and NEBNext Ultra II Q5 DNA Polymerase.

Techniques: Synthesized, Microarray, Amplification, Purification, Sequencing, Generated, Next-Generation Sequencing

Journal: Cell Reports Methods

Article Title: Retrospective cell lineage reconstruction in humans by using short tandem repeats

doi: 10.1016/j.crmeth.2021.100054

Figure Lengend Snippet:

Article Snippet: Sample specific barcoding PCR was performed using dual-index barcoding primers and NEBNext Ultra II Q5 DNA Polymerase.

Techniques: Recombinant, Purification, Sequencing, Software