Poly a mrna magnetic isolation module kit

Samples for RNA-Seq analysis included emasculated but unpollinated ovaries (E) as a control (absence of pollination, pollen tube growth and fertilization), and ovaries from IP6h, IP24h, DP24h, and XP24h style removal treatments. These samples were selected based on pollen tube growth observations; IP6h represented partial pollen tube growth (no penetration into the ovaries and hence no fertilization), IP24h represented full pollen penetration into the ovaries with fertilization, XP24h represented full pollen tube penetration without fertilization, while DP24h represented pollination only (no pollen tube growth and no fertilization). All samples were collected 48 h after pollination (2 DAA), and each sample contained 5 ovaries with three replications. Total RNA was extracted from the ovary samples using the RNeasy^® Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Treatment with DNase I (Nippon Gene, Tokyo, Japan) was carried out to remove genomic DNA contamination. mRNA was then purified from the total RNA using a poly(A) mRNA magnetic isolation module kit (New England BioLabs). The pure mRNA was used to construct paired-end libraries for Illumina using the Ultra™ II directional RNA library prep kit (New England BioLabs), and sequencing was performed on an Illumina NovaSeq 6000 platform (Illumina, Inc.).

Genomic DNA from the PA and PDX tumors and from the peripheral blood mononuclear cells (PBMCs) collected from 10 HGSOC patients were extracted using the QIAamp DNA Mini Kit (Qiagen, Cat#51306). DNA (0.3–0.5 µg) was sheared into 200- to 300-bp fragments using a Covaris^® M220 ultrasonicator followed by repair and 3’ poly-A tailing. Then, adaptors were ligated to both ends of the fragments and amplified via polymerase chain reaction (PCR). DNA libraries were generated with the xGen Hybridization and Wash Kit (IDT, Cat# 1080584), followed by PCR amplification. Total RNA of PA and PDX tumors from nine patients was extracted using TRIzol™ Reagent (Invitrogen, Cat# 15596026). RNA (0.1–1.0 µg) was used to generate libraries using the Poly(A) mRNA Magnetic Isolation Module Kit (NEB, Cat# E7490L) and NEBNext Ultra II RNALibrary Prep Kit (NEB, Cat# E7770L). The DNA and RNA libraries were sequenced on an Illumina NovaSeq 6000.