Additional file 24 of A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells
Creators: name: Bai, Yu
name: Caussinus, Emmanuel
name: Leo, Stefano
name: Bosshardt, Fritz
name: Myachina, Faina
name: Rot, Gregor
name: Robinson, Mark D.
name: Lehner, Christian F.
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 24: Fig. S14. Characterization of SR9 and SR9rg cells. (A-D) Identification of a functional target locus for RMCE in SR9rg cells. Whole genome sequencing was used for the characterization of the clonal SR9rg cell line. Hybrid paired reads, where one read aligns to genomic sequences and the mate read to pUC57-RMCE-target plasmid sequences were identified and used for mapping chromosomal insertions of pUC57-RMCE-target. An insertion that is competent for RMCE was identified on chromosome 3 L in an intron of the CG13288 gene. (A) The genomic region (dmel_r6.19, chr3L:5904000 to chr3L:5921000) with CG13288 as visualized by GBrowse 2.0 is displayed on top. The cumulative base coverage by hybrid paired reads mapping to this region is indicated by the blue bars below (from 0 to 55 reads/bp). Black arrows emphasize the distal clustering of the abrupt end of the alignments on either side, corresponding to the genome-plasmid junctions. Primers “L” and “R” (red arrows) with sequences close to the left and right ends of this genomic region were used for confirmation of the insertion by PCR (see panel D). (B) A linearized representation of pUC57-RMCE-target is shown on top. The grey bars below display the cumulative base coverage (from 0 to 51 reads/bp) by the hybrid paired reads mapping to the CG132888 region. Black arrows and dashed lines indicate breakpoints (abrupt clustered ends of read alignments). Primers “r” and “l” (red arrows) with sequences close to the right and left ends of the plasmid region were used for confirmation of the insertion by PCR (see panel D). (C) Proposed organization of pUC57-RMCE-target insertion in the CG13288 region. The plasmid pUC57-RMCE-target is inserted almost completely, starting from breakpoint “l” and extending to the breakpoint “r” (see panel B). The inserted plasmid region is flanked by a tandem duplication of the genome region between “L” and “R” (see panel A). Primers used for confirmation of the proposed organization are indicated (red arrows). (D) PCR assay for confirmation of the proposed organization of the pUC57-RMCE-target insertion in the CG13288 region and of its competence for RMCE. Genomic DNA isolated from the indicated cells was analyzed by PCR with the primer pairs indicated in the red boxes. Primer P in the last pair anneals to the pst_E1n region, which is only present after RMCE, i.e., only in the SR9rg &gt; pst_E1n cells. L = oEC264, R = oEC267, l = oEC263, r = oEC265, P = YB654. (E) Characterization of SR9 cells. These cells were obtained after transfection of S2R+ cells with the plasmid pMT-cas9-hygror schematically shown on top. For confirmation of CuSO4-inducible expression of cas9 in the SR9 cells, they were cultured in parallel with the parental S2R+ cells for 24 h without or with 500 μM CuSO4 as indicated. Total extracts were probed by immunoblotting with an anti-FLAG. Ponceau S staining (not shown) indicated comparable sample loading, as also indicated by a non-specific anti-FLAG band (*). The specific signal representing 3xFlag-nls-Cas9-nls (predicted molecular weight 164.67 kDa) is indicated as well (arrowhead).