GSM5931549: BV2_PBS rep3; Mus musculus; RNA-Seq
After exposure, the cells were lysed in 500 μL of cold cell lysis buffer containing 1% LiDS, 100 mM Tris-HCl pH 7.5, 500 mM LiCl, 10 mM EDTA, and 5 mM DTT (Shichino et al., 2019). The cell lysates were stored at -80 °C until analyses. Transcriptome libraries were prepared on cell lysate resolved in lysis buffer. PolyA RNAs were isolated using Dynabeads M-270 Streptavidin (Thermo Fisher Scientific) conjugated with biotin-3' WTA-EcoP-dT25 according to the previous report with some modifications (GSE110711). 100 ng of the whole-transcriptome library was subjected to fragmentation/end-repair/A-tailing using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs) with some modifications. The thermal cycling was performed as following condition: for 20 min at 32ºC, 30 min at 65ºC, and hold at 4ºC. Then, 1.25 μL of 1.5 μM IonA adapter was used for adapter ligation. Ligated products were purified by double size selection with 0.4× → 0.3× (final 0.7×) AmPure XP beads and eluted with 10 μL of nuclease-free water. The barcoding PCR was performed with 22.5 uL of barcoding mix [7.5 μL of the resulted eluates, 1 μM primers (IonA_[BC] and trP1), and 1× NEBNext Ultra II Q5 (New England Biolabs)], and the thermal cycling was performed as following condition: for 30 sec at 98°C, 9 cycles of 10 sec at 98°C and 75 sec at 65°C, followed by 5 min at 65°C, and hold at 4°C. Resultant products were purified twice by double size selection with 0.8× → 0.6 × (final 1.4×) AmPure XP beads and eluted with 12 μL of 10 mM Tris-HCl pH8.0. Size distribution of amplified products was analyzed by MultiNA system (Shimazu) and quantified using the KAPA library quantification kit (KAPA biosystems) with appropriate dilutions. Pooled libraries were sequenced by Ion Genestudio S5 sequencer (Thermo Fisher Scientific).