GSM8893547: POND-FMR1-A411T_rep2; Homo sapiens; RNA-Seq
Cell supernatant was clariﬁed by centrifugation at 3,000 g for 5 minutes, then filtrated through a cellulose acetate ﬁlter with 0.45 μm pore size (Corning). 200 μl of supernatant was mixed with 800 μl TRIzol reagent (Magen) and 1 μl diluted External RNA Controls Consortium (ERCC) synthetic spike-in RNAs (ThermoFisher). RNA was extracted and dissolved in 15-20 μl nuclease-free water. For POND-seq, 5 μl RNA was used for library construction with modified VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme). Typically, 5 μl RNA was mixed with 5 μl 2×Prime buffer (Vazyme) for fragmentation at 85℃ for 6 min. For ribosomal depletion, 5 μl RNA was mixed with 5 μl 2×Prime buffer (Vazyme) and 0.7 μl FastSelect reagent (Vazyme) for fragmentation at 85℃ for 6 min. Then 8 μl fragmentated RNA was used for first and secondary strand cDNA synthesis, followed by adapter ligation and PCR amplification (16-18 cycles) according to the manufacture's instruction manual. The DNA library was purified with VAHTS DNA Clean Beads (Vazyme) and dissolved in 20 μl nuclease-free water. The 150-nt paired-end sequencing was performed on Illumina NovaSeq 6000 (Novogene, China) or DNBSEQ-T7 platform (GenePlus, China). For FMR1 mutation screening, library construction was completed with VNL-96P (Vazyme) automated library preparation workstation. Briefly, 4 μl RNA was mixed with 4 μl 2×Prime buffer V2 (Vazyme) and 1 μl FastSelect reagent (Vazyme) for fragmentation at 85°C for 6 min. Then 8 μl fragmentated RNA was adopted for first and secondary strand cDNA synthesis, followed by adapter ligation and PCR amplification (17 cycles) according to VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina (Vazyme). The NGS sequencing was performed on DNBSEQ-T7 platform (GenePlus). For cellular RNA-seq, the total RNA of HEK293T, Huh-7 and A549 were extracted with TRIzol reagent (Magen) and dissolved in nuclease-free water. 500 ng total RNA was used for library construction with VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme). The DNA library was purified with VAHTS DNA Clean Beads (Vazyme) and dissolved in 20 μl nuclease-free water. The 150-nt paired-end sequencing was performed on Illumina NovaSeq 6000 (Novogene) or DNBSEQ-T7 platform (GenePlus).