Dna clean concentrator 5 kit

E. coli K12 CGSC4401 containing plasmid pDSW1728-hygR served as the donor strain. E. coli BW25113 strains containing base editor, hygR Rptrs and recording plasmids served as the recipient strains. The donor strain and recipient strains were grown overnight at 220 r.p.m. and 37 °C in LB + Hyg and LB + Cm + Amp + Kan liquid media, respectively. The donor strain was 1:50 back-diluted into 100 ml LB + Hyg medium for 3 h growth at 220 r.p.m. and 37 °C. The recipient strains were 1:10 back-diluted into 25 ml LB + Cm + Amp + Kan + IPTG + l-arabinose medium for 3 h growth at 220 r.p.m. and 37 °C. After 3 h of growth, both donor strain and recipient strains were gathered, washed twice and then resuspended in 1× PBS. The mixtures of donor and recipient strains in a ratio of 10:1 were spotted onto LB + Cm + Amp + Kan + Hyg + IPTG + l-arabinose plates for 2–3 days of growth at 37 °C. As a negative control, the same amounts of recipient strains were spotted onto LB + Cm + Amp + Kan + IPTG + l-arabinose plates for 2–3 days of growth at 37 °C. Colony PCR was performed to amplify the editing region using primers CJpr0001 and CJpr0002. The PCR products were cleaned up using the DNA Clean & Concentrator-5 kit (Zymo Research, catalog no. D4013) and then sent for Sanger sequencing with primer CJpr0001. The web tool EditR v.1.0.10 was used to evaluate the editing efficiency^{65 (link)}.

The oligopaint probes were cleaned using the DNA Clean & Concentrator‐5 kit (Zymo Research, DCC‐5). 50 μl PCR product was mixed with 350 μl Zymo DNA binding buffer, transferred to a Zymo DCC‐5 column, and spun at 16,000 g for 1 min. Afterward, 200 μl DNA Wash Buffer was added to the column and centrifuged at 16,000 g for 1 min. The wash step was repeated. The flow‐through was discarded, and the column was spun at 16,000 g for 1 min. The column was transferred to a clean 1.5‐ml tube, and 11 μl ddH₂O was added. The samples were incubated at room temperature for 1 min and centrifuged at 16,000 g for 1 min.

In situ RNA probes for olig2, mnx1, myt1a, myt1b, insm1a, and tac1 were generated by a plasmid-free approach as reported previously (54 ). Primers used for probes were listed in Table S1. cDNAs from 2 and 3 dpf embryos were amplified with related primers, and PCR products were purified with the Zymo Research DNA Clean & Concentrator-5 kit. T7 polymerase was used to make digoxigenin-labeled antisense probes for in situ hybridization. Whole-mount in situ hybridization and fluorescence in situ hybridization followed by immunostaining of embryo sections were performed as previously described (53 (link)).

The amplified oligopaint library was cleaned using DNA Clean & Concentrator‐5 kit (Zymo Research, DCC‐5). 25‐μl PCR product was mixed with 175 μl Zymo DNA binding buffer, transferred to a Zymo DCC‐5 column, and spun at 16,000 g for 1 min. Next, 200 μl DNA wash buffer was added to the column and centrifuged at 16,000 g for 1 min. The wash step was repeated. The flow‐through was discarded, and the column was spun at 16,000 g for 1 min once more. The column was transferred to a clean 1.5‐ml tube, and 30 μl ddH₂O was added. The column was incubated at room temperature for 1 min and centrifuged at 16,000 g for 1 min. The concentration was measured using a NanoDrop device.

The 16S rRNA gene was PCR-amplified using Econotaq and primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r primer (5′-TACCTTGTTACGACTT). The size of the PCR products was determined using gel electrophoresis, then cleaned using DNA Clean & Concentrator-5 Kit (Zymo), and sequenced using Eton Bioscience, Inc. 16S sequences were aligned using MUSCLE [39 (link)] with default parameters, and trimmed in Jalview v2.11.1.6 [42 (link)]. Publicly available 16S sequences were obtained from the NCBI database as references. A maximum likelihood tree was constructed using the IQtree webserver [43 (link)], using the best-fit model K2P + G4 automatically chosen according to Bayesian Information Criterion by ModelFinder [44 (link), 45 (link)], with 1,000 bootstraps [46 (link)]. The tree was visualized using the Interactive Tree of Life (iTOL) v.4 webserver (https://itol.embl.de/) [47 (link)].

To identify the editing window for Sth1Cas9n base editor, three sgRNAs were designed with poly Cs located in different positions of the guide sequence. Colonies of E. coli containing the recording machinery were inoculated into LB + Cm + Amp + Kan liquid medium. Cultures were then grown overnight at 220 r.p.m. and 37 °C. The overnight cultures were back-diluted 1:20 into 5 ml of LB + Cm + Amp + Kan + IPTG + l-arabinose liquid medium for 8 h induction at 220 r.p.m. and 37 °C. Then 2 ml of induced cultures were collected for plasmid extraction using ZR Plasmid Miniprep-Classic kit (Zymo Research, catalog no. D4016). Next, 50 ng of the extracted plasmid was used as a template to amplify the edited region using primers CJpr0001 and CJpr0002. The PCR products were purified with the DNA Clean & Concentrator-5 kit (Zymo Research, catalog no. D4013) and then sent for Sanger sequencing with primer CJpr0001. The web tool EditR v.1.0.10 (https://moriaritylab.shinyapps.io/editr_v10/) was used to evaluate the editing efficiency^{65 (link)}.

All the first-strand synthesis reactions were performed in 10 μL total volume using various amounts of RNA (50 pg–2 μg), 1 μL of 10 μM barcoded oligo-dT (BU3, Microsynth, for the list of oligos used see Additional file 2: Table S6 and S7), and either 0.125 μL of Maxima H Minus Reverse Transcriptase (MMH, ThermoFisher Scientific, #EP0753) or 0.25 μL Superscript II (SSII, Invitrogen, #180640). The reactions followed by the PCR pre-amplifications were complemented with 1 μL of 10 μM template switch oligo (TSO, IDT). RNA, BU3 primers, and 1 μL dNTP (0.2mM) were mixed together in a PCR plate, incubated at 65 °C for 5 min and then put on ice. The TSO, RT buffer (including 1 μL of DTT for the Superscript II protocol), and RT enzymes were added to each well, and the plates were incubated at 45 °C for 90 min for the Maxima protocol or 42 °C for 50 min followed by inactivation at 70 °C for 15 min for the Superscript II protocol. After RT, all the wells were pooled together and purified using the DNA Clean & Concentrator-5 kit (Zymo Research, #D4014) with 7× DNA binging buffer and single column. After elution with 20 μL of nuclease-free water, the samples were incubated with 1 μL Exonuclease I (NEB, #M0293) and 2 μL of 10× reaction buffer at 37 °C for 30 min, followed by enzyme inactivation at 80 °C for 20 min.