RNA-seq of Schmidtea mediterranea; isolated pre-pharyngeal region
Planarians used for RNA extraction were 5-7 mm in length. DMSO control, 10 M DPI, and 15 M DPI samples were pre-treated 24 hours prior to amputation and RNA extraction. For control samples, the pre-pharyngeal region was isolated and immediately submerged in liquid nitrogen. For all other conditions (untreated decapitation R-wounds, untreated slit H-wounds, DMSO-treated R-wound controls, and DPI-treated R-wounds), animals were either amputated anterior to the pharynx or an incision (slit) was made in the pre-pharyngeal region. Pre-pharyngeal regions were pooled from n = 40 animals for each biological replicate, and each condition was repeated n = 3 times. Animals were incubated in either DMSO, DPI, or untreated media for 45 minutes at 20C after injury. The wound site was isolated at 45 minutes and tissues were immediately frozen in liquid nitrogen. Frozen tissue was ground to a fine powder with mortar and pestle and resuspended with lysis buffer from the Qiagen QIAshredder assay kit (Cat. No. 79656). Tissue lysis was performed according to the manufacturers protocol.Total RNA was extracted using the Qiagen RNeasy Plus Micro Kit (Cat. No. 74034) following the manufacturers protocol. RNA purity and concentration was assessed using both Invitrogen Qubit RNA High Sensitivity Assay (Cat. No. Q32852) on a Thermo Fisher Qubit Fluorometer and Agilent RNA ScreenTape (No. 5067-5576) on an Agilent TapeStation 2200 according to the manufacturers suggested protocols. Samples were stored in nuclease-free water at -80C prior to library preparation. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. M-MuLV Reverse Transcriptase (RNase H-, Promega) was used for first-strand cDNA synthesis, while DNA Polymerase I and RNase H (Promega) were used for second strand synthesis. Library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments 370~420 bp in length. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Purified PCR products (AMPure XP system) and library quality were assessed using an Agilent Bioanalyzer 2100 system. Index-coded samples were clustered with a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturers protocol. Paired-end sequencing reads of 150 bp were generated from library preparations using an Illumina Novaseq 6000 platform.                                                           An index of the reference genome (Schmidtea mediterranea S2F2 genome; NCBI Assembly GCA_000691995.1) and alignment of clean paired-end reads was performed using Hisat2 v2.0.5. Mapped reads were assembled by StringTie (v1.3.3b) using a reference-based approach and gene expression levels (Fragments Per Kilobase of transcript per Million mapped reads, FPKM) were quantified with featureCounts v1.5.0-p3. Differential expression analysis was performed using the DESeq2 (1.20.0) and significance values (p  0.05) for normalized expression values were adjusted for false discovery rate using the Benjamini and Hochbergs approach.