Pmaxgfp vector

The pSpCas9(BB)-2A-GFP (PX458) plasmid was a gift from Feng Zhang^{49 (link)} (Addgene plasmid # 48138). The pmKaxxte plasmid (now available as Addgene plasmid #113630), used to check gRNA efficiency, was generated using the pmaxGFP vector (Lonza) as a backbone and the mKate2.5-C1 — a gift from Michael Davidson (Addgene plasmid # 54828) — to generate overlapping insert fragments and a multiple cloning site.
The RFP2GFP-ATM construct was generated by inserting RFP sequence from mKate2.5-C1 into the pmaxGFP vector between KpnI and AgeI sites. The sequence encoding the ATM exon20-intron20-exon21 was PCR amplified from 293 T derived genomic DNA using primer sequences in Table S1 and cloned into the vector’s ORF between RFP and GFP tags using HindIII and SalI enzymes.

A codon optimised sequence with identical coding potential but altered RNA sequence to LINC01013ORF was cloned into expression vector pcDNA3.1 (Addgene) and expressed under control of a CMV promoter/enhancer. Control vector contained eGFP also expressed from CMV promoter/enhancer in the pMaxGFP vector (Lonza).

Wild-type prkar2b subunit of bovine PKA and mutant prkar2b (harboring mutations that ablate all potential miR-34c seed sites) were synthesized (GenScript) and cloned into pmaxGFP vector (Lonza). Virulent T. annulata-transformed macrophages were transfected with plasmids expressing wild-type or mutant PRKAR2B with GFP fused to the N termini. The transfection rate (efficiency) was measured, and in 3 independent transfections, the efficiency averaged 19%. Therefore, to obtain a high percentage of transfected cells, 24 h posttransfection, GFP-expressing cells were separated using fluorescence-activated cell sorting (FACS) with the MoFlo Astrios instrument (Beckman). Following sorting, total RNA was prepared as described above.

The NIH-3T3 cells were plated on coverslips and co-transfected with the BRCA2-CT cDNA constructs and pmaxGFP^® Vector (Lonza) (10:1 rate). Twenty-four hours after the transfection, the cells were irradiated with 5 Gy. The cells were fixed 30 min, 12 h, 24 h and 36 h after irradiation with 4% PFA for the immunofluorescence assay. A non-irradiated control was fixed for each experimental point. The coverslips were blocked and permeabilized using 10% FBS, 1% BSA, and 0.2% Triton X-100 in PBS 30 min at room temperature and incubated with anti-phospho-Histone H2A.X (Ser139) (1:300, Millipore, MA, USA) overnight at 4 °C. After washing, the coverslips were incubated with the Alexa Fluor 594-conjugated secondary antibody (Molecular Probes) for one hour at room temperature. DAPI (Molecular Probes) was used to stain the nuclei. Fluorescence was observed with the Leica DMS 4000B microscope (Leica, Wetzlar, Germany) with a 20× objective. DNA damage recovery was quantified as the number of γ-H2AX-positive cells on the total number of GFP-positive cells. Ten fields for each sample were analyzed with the ImageJ software, and the number of γ-H2AX-positive cells was calculated from a minimum of 500 cells per dose/time point.

pX330-U6-Chimeric_BB-CBh-hSpCas9 was purchased from Addgene, (Cambridge, MA, USA). The single guide RNA sequences targeting the Nrp2, Dcstamp, and Nfatc1 eRNA regions were designed using Optimized CRISPR Design³⁹ . The guide sequences are listed in Supplementary Table S3. The universal negative control containing a scrambled sequence was 5′-GCACTACCAGAGCTAACTCA-3′^{40 (link)}. The targeting vector of each eRNA region was transfected together with the pmaxGFP^® vector (Lonza, Basel, Switzerland) into RAW 264.7 cells using the Amaxa Cell Line Nucleofector Kit V (Lonza). The transfected cells were cultured for 2 days, and single-cell sorted using an SH800 cell sorter (Sony, Tokyo, Japan). The knockout clones used in these studies were validated by sequencing.