Cell line 96 well nucleofector kit sf

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).

LCLs were seeded at a density of 550,000 cells/mL 24 hours before nucleofection. Amaxa's Cell Line 96-well Nucleofector Kit SF (Lonza Inc, Basel, Switzerland) was used to perform the transfection. Cells were centrifuged at 90 g for 10 minutes at room temperature and resuspended at a concentration of 1,000,000 cells in 20 µL of SF/supplement solution (included in SF Kit Lonza Catalog #V4SC2096) and 2 µM final concentration of AllStars negative Control siRNA labeled with AlexaFluor488 (Qiagen Inc., Valencia, CA) or a pool of siRNA (Qiagen) (See Table S1). The cells were nucleofected using Amaxa's DN-100 program. Cells were allowed to rest for 10 minutes before the addition of pre-warmed (in 37° water bath for a minimum of 20 minutes) RPMI media and then another 5 minutes after the addition of warm RPMI media. Cells were then plated for protein measurements and drug treatments. Cells were harvested at 24 and 48 hours post-nucleofection for protein measurement. Drug treatment was done 18 hours following transfection for cell survival measurement and 24 hours after transfection for apoptosis measurement. Apoptosis was measured as described above, whereas cell survival was measured as described above for cisplatin and using Cell-Titer Glo (Promega) for paclitaxel. Each experiment was done twice, with two independent transfections.

To generate CREBBP-KO, CREBBP-R1446C, KMT2D-KO, and double loss-of-function isogenic OCI-Ly7 cells, ribonucleoprotein (RNP) complex containing Alt-R recombinant S.p. HiFi Cas9 Nuclease (IDT; 1081061), Alt-R CRISPR-Cas9 tracrRNA (IDT; 1072534), and Alt-R CRISPR-Cas9 crRNA targeting CREBBP exon 26 or KMT2D exon 4, were assembled in vitro following the manufacturer’s protocol and delivered into OCI-Ly7 cells by electroporation using SF Cell Line 96-well Nucleofector Kit (Lonza; V4SC-2096). ssODN for CREBBP R1446C mutagenesis was added along with RNP complex during electroporation as needed. Single cells were then seeded in 96-well plates by serial dilution. Clones were screened by Sanger sequencing of PCR amplicons and immunoblot. crRNA, ssODN and genotyping primer sequences can be found in Supplementary Data 7.

To knock down a target gene in CH12F3-2A cells, we introduced 40 pmoles of chemically modified Stealth siRNA oligonucleotide (Thermo Fisher) into approximately 1 × 10⁶ cells by electroporation. The 96-well Lonza Nucleofector electroporation system and the SF Cell Line 96-well nucleofector kit (Lonza #V4SC-2096) were used to introduce siRNA or plasmid into CH12 F3-2A cells using the Nucleofector program # CM-150. One day later, the cells were stimulated by CIT and cultured for another 1–2 days before harvest and downstream analysis. For the CSR complementation assay, siRNA and 1–1.5 μg of EGFP fused mBrd2^R-MF construct (Figure 1G) were co-electroporated into CH12F3-2A cells. To induce CIT-independent CSR in CH12F3-2A cells, Sμ- and Sα-specific CRISPR/Cas9 constructs were co-electroporated with siControl or siBrd2 as needed. To deliver siRNA into the primary B cells, ∼6–7 × 10⁵ LPS preactivated B cells mentioned were subjected to electroporation using a mouse B-cell Nucleofector Kit and the program # 96-DI-100. Supplementary Table S2 contains necessary information on the siRNAs used.