Pe150

Nuclei were extracted from cortex homogenates and incubated with RNaseA (Sigma R6513) at 37 ℃ for 15 min, followed by proteinase K (Sigma P2308) incubation at 52 ℃ overnight. The samples were mixed with phenol/chloroform (1:1) (Solarbio P1021), and centrifugated. The supernatant was collected and then precipitated by isopropanol. The product was washed in 70% ethanol, air dried, and dissolved by buffer EB (Qiagen 19086). A total of 1 μg DNA was used for RRBS library preparation and sequenced with Novaseq set paired-end, 150 bp (PE150) (Novogene, China). Briefly, unmethylated lambda DNA was added into the genomic DNA (gDNA) and incubated with MspI enzyme to obtain 200 to 1000 bp fragments. The DNA fragments were then converted by bisulfite using the EZ DNA Methylation-Gold Kit (Zymo Research, United States).

RNA samples were extracted from fresh tumor biopsies from the MAP model and pancreatic ductal adenocarcinoma model: Kras^G12D; p53^Lox/lox; Ptf1a-Cre (KPC) previously described^{60 (link)}. Briefly, biopsied tumors were homogenized with TRIzol reagent (Life Technologies) and centrifuged at 16,000 RPM for 15 min at 4 °C. The samples received additional precipitation with a 1:2 chloroform:isoamyl alcohol pH 8.0 solution and centrifuged at 12,000 RPM for 15 min to promote phase separation of RNA. Three layers formed from this precipitation: an upper aqueous phase (containing RNA), a white interphase (containing DNA), and a lower organic phenol:chloroform phase (consisting of proteins). The upper aqueous phase was collected and mixed 1:1 with 100% ethanol. The Direct-zol RNA miniprep kit (cat. no. R2050) was utilized. RNA samples were sequenced at 20 M reads, PE150 (Novogene).

For the metagenomes newly presented in this study Spanish lakes and reservoirs were sampled in two different seasons (winter-mixed and summer-stratified periods) and for each lake/season representative samples corresponding to the epilimnion, hypolimnion and DCM (for the summer period) were obtained. This allowed us to monitor the abundance of α- and β-cyanobacteria at different times of the year. No blooms of β-cyanobacteria were detected in any of the Spanish lakes from which metagenomes were obtained. Further details of sampling metadata, including the depth and sample location are given in Supplementary Dataset 1. Pelagic water samples from the different Spanish lakes (Lakes La Cruz, Cardenillas, Arcas and El Tobar, and Tous, Loriguilla, Amadorio and Benageber reservoirs) were obtained through a 3-year sampling campaign. Briefly, 20 l water were sequentially filtered through 20, 5 and 0.22 µm pore size filters and DNA extracted with CTAB-lysis buffer followed by phenol-chloroform-isoamyl alcohol extraction [74 (link)]. We exclusively sequenced (NovaSeq (Illumina, USA) PE150, Novogene UK) the small plankton fraction that passed through the 5 µm pore size filter but which was retained on the 0.22 µm pore size filter. Approximately 15 Gb/output (ca. 100 million reads) were obtained for each metagenome.