GSM8582112: B73_prophase_I_male_germ_cell_RNA_rep1; Zea mays; RNA-Seq
Total RNA was isolated from embryos, anthers and male germ cells using TRIzol (Takara, 9109). MgCl2 (final concentration: 0.2 M) was added to prevent loss of sRNAs with low GC content (Kim et al., 2012). NaAc (pH 5.2) and glycogen were added to promote RNA precipitation. The extracted RNA was stored at −80°C in LoBind tubes (Eppendorf, 022431021). Libraries for RNA sequencing were prepared using the Smart-seq method with minor modifications, as previously described (Jiang et al., 2020; Picelli et al., 2014). In brief, total RNA was reverse transcribed and cDNA second strand was subsequently synthesized using SuperScript II reverse transcriptase (Invitrogen,18064-14) and template-switching oligos. After PCR preamplification and purification, the products were fragmented using TruePrepTM DNA Library Prep Kit (Vazyme, TD501). The fragments were amplified with index-containing primers and the 300–800-bp products were selected with AMPure XP beads. Libraries were pair-end sequenced on with Illumina Nova platform by Annoroad Gene Technology (Beijing).