Cell line nucleofector kit 5

OSCs (a gift from Mikiko Siomi) were cultured as described (Niki et al., 2006 (link); Saito, 2014 (link); Saito et al., 2009 (link)). S2 cells (Thermo Fisher Scientific; R69007) were cultured in Schneider’s Drosophila medium (Gibco) supplemented with 10% Heat-Inactivated FBS (Sigma). Cell identity was authenticated in-house by high-throughput sequencing approaches and cell lines regularly tested negative for mycoplasma contamination in-house. Knockdowns in OSCs were performed as described previously (Saito, 2014 (link)). A list of siRNA sequences is provided in Supplementary file 3. Transfections in OSCs were carried out using either Xfect (Clontech) or the Cell Line Nucleofector kit V (Amaxa Biosystems) with the program T-029, as described (Saito, 2014 (link)). Rescue experiments in OSCs were carried out as described (Fabry et al., 2019 (link); Munafò et al., 2019 (link)). S2 cells were transfected with the Cell Line Nucleofector kit V (Amaxa Biosystems) using the program G-030, as described (Batki et al., 2019 (link)). Stable cell lines were generated by co-transfection of the HA-ZsGreen reporter plasmid with a plasmid expressing a puromycin resistance gene under the control of the metallothionine promoter. Cell with reporter integrations were selected with puromycin treatment until stable lines were generated, with cell lines subsequently being cultured without puromycin.

For siRNA transfection into OSCs, 200 pmol siRNA duplex was nucleofected into 3.0 × 10⁶ cells using the Cell Line 96‐well Nucleofector Kit SF (Lonza) and program DG150 of the 96‐well Shuttle Device (Lonza) or Cell Line Nucleofector Kit V (Lonza) and program T‐029 of the Nucleofector II Device (Lonza). The siRNAs were transfected twice for 4‐day KD and once for 2‐day KD. Piwi‐KD was performed for 4 days, while Lam/LamC‐KD was performed for 2 days due to the severe damage caused to cells. siRNA sequences are listed in Table EV2. All of the siRNAs were tested for their efficiency to silence target genes in the previous study (Iwasaki et al, 2016 (link); Murano et al, 2019 (link)). Co‐transfection of siRNA and plasmid DNA was performed using the Cell Line Nucleofector Kit V (Lonza) and program T‐029 of the Nucleofector II Device (Lonza).

1×10⁶ K562 or KCL22 cells were transiently nucleofected with 1 µg of each minigene using the Cell Line Nucleofector Kit V (Lonza). RNA was extracted from these cells after 24 hours. For knockdown studies, siRNAs were synthesized either from Integrated DNA Technologies or from Ambion. The siRNA sequences were: control siRNA (5′-GGUCCGGCUCCC-CCAAAUC-3′) [26] (link), PTBP1 siRNA 1 (5′-CUUCCAUCAUUCCAGAGAA-3′) [26] (link),
PTBP1 siRNA 2 (5′- CAAAGCCUCUUUAUUCUUU-3′), PTBP1 siRNA 3 (5′-CAGUUUACCU-GUUUUUAAA-3′) hnRNP H (siH1: 5′-GAAGCAUACUGGUCCAAAU-3′; siH2: 5′-GGAUUU-GGGUCAGAUAGAU-3′), hnRNP F (siF1: 5′-GCAGCACAGAUAUAUAGAA-3′; siF2: 5′-CGA-CCGAGAACGACAUUUA-3′) and hnRNP C (siC1: 5′-CAACGGGACUAUUAUGAUA-3′;
siC2: 5′-GAUGAAGAAUGAUAAGUCA-3′; siC3: 5′-ACACUCUUGUGGUCAAGAA). Knockdowns were performed using siRNAs at 100 nM concentration on 1×10⁶ K562 cells using the Cell Line Nucleofector Kit V (Lonza).