deadenylase  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: A low-bias and sensitive small RNA library preparation method using randomized splint ligation

doi: 10.1093/nar/gkaa480

Figure Lengend Snippet: Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.

Article Snippet: These reactions were incubated in a thermocycler at 25°C for 1 h. Following ligation 2.5 units of lambda exonuclease (NEB M0262) and 25 units of 5′ deadenylase (NEB M0331) were added and the reactions were incubated for 15 min at 30°C, 15 min at 37°C and 5 min at 75°C.

Techniques: Ligation, Synthesized, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting

doi: 10.1101/295253

Figure Lengend Snippet: Principle and technical parameters of TAC-seq. ( A ) Schematic diagram of the assay to detect specific mRNA or cell-free DNA. Target-specific DNA oligonucleotide detector probes hybridize under stringent conditions to the studied cDNA or cfDNA. Both detector oligonucleotides consist of a specific 27-bp region (green), 4-bp unique molecular identifier (UMI) motif (NNNN) and universal sequences (purple and orange). The right detector oligonucleotide is 5’ phosphorylated. After rigorous hybridization, the pair of detector probes is ligated using a thermostable ligase under stringent conditions. Next, the ligated detectors complexed with the target region are captured with magnetic beads and PCR amplified to introduce sample-specific barcodes and other common motifs that are required for single-read NGS. ( B ) Spearman correlation analysis of the input and detected ERCC synthetic spike-in mRNA molecules at UMI threshold 4 (UMI=4). UMI threshold is defined as the number of detected unique UMI sequences. For example, UMI=4 indicates that a certain UMI motif is detected at least four times. UMIs are valuable only if the number of UMI combinations (8-bp UMI provides 65,536 variants, for example) is substantially larger than the sum of the target molecules in the studied sample. ( C ) Bar plot of Spearman’s correlation analysis of the ERCC input and detected molecules at different UMI thresholds. ( D ) Reproducibility of seven technical ERCC replicates (seven different icons on plot) of 22 spike-in molecules at UMI=4.

Article Snippet: After ligation, the free ligation adapter was removed by adding 0.5 µl 5’-Deadenylase (25 U/µl, NEB) and 0.5 µl Lambda exonuclease (5 U/µl, NEB) and incubated 10 min at 37°C, followed by 10 min at 75°C. cDNA was synthesized after adding 0.4 µl 100 mM DTT (Invitrogen), 0.4 µl 2 M KCl (Sigma-Aldrich), 0.4 µl 10 mM dNTPs (Thermo Fisher), 0.4 µl RNase inhibitor (Thermo Fisher), 0.2 µl 10 µM micro RT biotin primer and 0.2 µl Maxima H Minus Reverse Transcriptase (200 U/µl, Thermo Fisher) which were mixed into one 2 µl master mix. cDNA incubation was carried out for 15 min at 50°C, followed by 5 min at 80°C.

Techniques: Hybridization, Magnetic Beads, Polymerase Chain Reaction, Amplification, Introduce, Next-Generation Sequencing