Amaxa nucleofector unit

To generate SETD2 KO cell lines (HBL1, OCI-Ly7, SU-DHL-4, SU-DHL-2), parental cells were transduced with lentivirus expressing doxycycline inducible Cas9 and blasticidin resistance gene (Addgene, #83481) followed by 5 days of blasticidin selection. Cas9 expressing cells were transduced with lentivirus expressing a SETD2 specific sgRNA (SETD2G#1: 5’ AAA GAA ACA ATA GTA GAA GT 3’; SETD2G#2: 5’ AAT CTG ATG AAG ATT CTG TA 3’) and GFP (Addgene, #57822). 4 days post doxycycline induction, GFP⁺ cells were single cell sorted into 96 well plates and allowed to grow for at least two weeks. For RIVA, parental cells were electroporated with Amaxa Nucleofector Unit and the SF Cell line 4D-Nucleofector X kit (Lonza, PBC2–22500) to incorporate a recombinant Cas9 nuclease (Alt-R S.p. Cas9 Nuclease V3, Integrated DNA Technologies, 1081058), a SETD2 targeting Alt-R CRISPR-Cas9 crRNA (SETD2G#1: 5’ AAA GAA ACA ATA GTA GAA GT 3’; SETD2G#2: 5’ AAT CTG ATG AAG ATT CTG TA 3’ Integrated DNA Technologies) and Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies, 1075927) using manufacturer’s protocol. Forty-eight hours after electroporation, ATTO550+ single cells were sorted into 96 well plates and allowed to grow for at least two weeks. Clones were screened by PCR amplification of a 500bp region encompassing the CRISPR-Cas9 cleavage site and verified by sanger sequencing at GENEWIZ.

Stem cell cultures and neuronal differentiation were conducted as previously described ^{17 (link), 19 , 26 (link)}. Unless otherwise indicated human embryonic stem cell (hESCs) line H1 ^{27 (link)} was used to differentiate human cortical neurons for most of the experiments. In addition, human induced pluripotent stem cell (iPSC) line 20B ^{28 (link)} was also used to differentiate human cortical neurons for studies that analyzed gene expression for CREB pathway components. For extensive methods used see supplementary methods section. shRNA lentiviral constructs targeting ASH1L, containing a puromycin resistance cassette were purchased from Sigma (Supplementary Table S10). For each reaction, 400,000 human cortical neurons ^{17 (link)} between days 28 to 30 were nucleofected with control or ASH1L shRNAs constructs using the X-unit of the Amaxa nucleofector unit (Lonza, catalog # AAF1002X) per manufacturers protocol. Neurons were subjected to puromycin selection a day after nucleofection and were harvested 72 hours (hrs.) post puromycin treatment unless otherwise indicated. A detailed protocol is included in the supplementary methods.