Micro plus kit

Total RNA was extracted from the caudal region (below forelimb) of E9.5 Foxa2^mcm; Rosa26^LSL-NICD using a Qiagen Micro Plus kit. cDNA was synthesised using SuperScript III reverse transcriptase (Life Technologies). qRT-PC was performed with Power SYBR Green Master Mix (Life Technologies) and reactions measured in a C1000 Thermal Cycler (Bio-Rad) under the following conditions: 95°C for 5 min, 40 cycles 95°C for 15 s and 60°C for 1 min. Ptch1, Gli1 (Han et al., 2009 (link)), Hes1 (Li et al., 2012 (link)) and cHairy2 primers (F: 5′-CCGTACCCTGCAAGCCAGGTG-3′, R: 5′-GCCCATCA-GAGGCAAGCAGCA-3′) were described previously and normalised against β-actin (Ferjentsik et al., 2009 (link); Ribes et al., 2010 (link)) using the Pfaffl equation (Pfaffl, 2001 (link)).

Freshly harvested embryos were frozen in OCT medium (Sakura Finetek), sectioned at 10 μm thickness and transferred to PEN slides (Ziess). To fix and stain the slides, they were dipped in 95% ethanol for 2 min to fix the samples, stained with crystal violet stain (3% in ethanol) on ice. The slides were then rigorously washed in 2 X 70% ethanol for 30-40 s to remove the OCT and dehydrated in 100% ethanol for 2 min. For control and Etv mutant embryos, the lens tissue was micro-dissected from the transitional zone using Laser capture microscope (Zeiss AxioObserver.Z1 inverted microscope). The RNA was extracted using Qiagen Micro Plus kit. Conversion to cDNA and amplification were performed using Clontech SMART-seq v4 Ultra low input RNA kit and the cDNA library construction was performed using Nextera XT DNA library preparation kit by the core facility at Columbia university prior to RNA sequencing. The RNAseq data are available from the GEO repository (GSE137215). Normalization and differential gene expression analysis for RNA-seq data were performed using De-Seq package in R studio. The GSEA analysis was performed using JavaGSEA desktop software from Broad Institute.

RNA was extracted from single embryos using a Microplus kit (Qiagen) according to manufacturers’ instructions. 500 ng of RNA was used for cDNA synthesis using a SuperScriptIII First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen). qPCR was performed in triplicate on a 7900 Fast Machine (Life Technologies) using Fast SYBR Green Mastermix (Life Technologies) with 20 ng cDNA and 500 nM forward and reverse primers in a final 20 μl reaction volume (see S4 Table for primer sequences). Quantitation was relative to Hprt and fold changes were calculated using the ΔΔC_T method (7500 Software v2.0.6., Life Technologies).

APOBEC-YTH and APOBEC-YTH^mut were purified and in vitro DART-Sanger sequencing assays were performed as previously described (Tegowski et al., 2022 (link)) with minor modifications. Briefly, total RNA was isolated from adult heads with TRIzol (Invitrogen) and treated with DNase I (NEB). RNA was isolated once more with TRIzol (Invitrogen) to remove DNase I and DNase I Buffer (NEB). Next, 200 ng of purified RNA from Drosophila heads was incubated with 1000 ng of purified DART protein in DART buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl, 0.1 M ZnCl₂) and 1 µL of RNaseOUT (Invitrogen) in a total volume of 200 µL for 4 hr at 37°C. RNA was isolated with the QIAGEN Plus Micro Kit (QIAGEN) and stored at –80°C before being thawed for downstream Sanger sequencing analysis. cDNA was made using iScript Reverse Transcription Supermix (Bio-Rad). PCR amplification of Sxl pre-mRNA was carried out with Phusion High Fidelity PCR Kit (NEB). The resulting PCR product was PCR-purified using the QIAGEN PCR Purification Kit (QIAGEN). Samples were submitted for Sanger sequencing (McLabs) and %C-to-U editing was quantified using EditR software (Kluesner et al., 2018 (link)).