Protocol for detecting genomic insulators in Drosophila using insulator-seq, a massively parallel reporter assay

Abstract

Genomic insulators are DNA elements that prevent transcriptional activation of a promoter by an enhancer when interposed. We present a protocol for insulator-seq that enables high-throughput screening of genomic insulators using a plasmid-based massively parallel reporter assay in Drosophila cultured cells. We describe steps for insulator reporter plasmid library generation, transient transfection into cultured cells, and sequencing library preparation and provide a pipeline for data analysis. For complete details on the use and execution of this protocol, please refer to Tonelli et al.¹.

Keywords: Gene Expression; Model Organisms; RNA-seq; Sequencing.

Graphical abstract

Figure 1

Barcode oligo design The parts of the insulator-seq barcode oligos are shown. The color code used for gene-identifying sequences was kept in all figures.

Figure 2

Overview of the major protocol steps The flowchart shows the main steps and the substeps of the insulator-seq protocol.

Figure 4

Agarose filter preparation and use (A) Agarose filter with a pipette tip inside after agarose hardened. (B) Empty well created by removing the pipette tip after agarose hardened. (C) Sample loaded onto the agarose filter. Note the air pocket under the sample. (D) Same as C, but the sample was colored to help visualize the correct position of the sample in the filter. We do not recommend staining the sample for the experiment.

Figure 5

Tagmentation PCR on the gel Tagmentation PCR after 16 cycles of amplification visualized on a 1% agarose-TBE gel after 30 min of electrophoresis. DNA size ladder is labeled. Gel fragment outlined by the red dashed lines were excised from the gel and purified.

Figure 3

Insulator-seq reporter library cloning (A) Barcode oligo pool is amplified by PCR (step 1a). (B) Amplified barcode oligo pool and vector (insulator-seq barcode vector; Addgene 221458) are digested with BsaI (step 1b). (C) BsaI-digested barcode oligo pool and vector are ligated, preserving BsaI-recognition site (step 1c). (D) Result of cloning step 1: single barcode plasmid (step 1d). (E) Single barcode plasmid pool is digested with BsaI (step 2a). (F) BsaI-digested barcode oligo pool and BbsI-digested single barcode plasmid pool are ligated. (G) Result of cloning step 2: Double barcode plasmid pool. (H) 3′UTR-Enhancer-Neutral DNA-3′UTR sequence (Addgene 221459) is inserted between the double barcodes by BbsI-mediated Golden Gate assembly (step 3a). (I) Result of cloning step 3: double barcodes separated by the enhancer, neutral DNA, parts of 3′UTR sequences. (J) Double barcode and enhancer insert pool is inserted between the reporter genes (Addgene 221460) by BsaI-mediated Golden Gate assembly (step 4a). (K) Result of cloning step 3: insulator-seq acceptor library. (L) Insulator-seq insert is generated by tagmenting DNA of the locus of interest (step 5). (M) Insulator-seq insert is generated by PCR-amplifying designer oligo pool library (step 6). (N) Insulator-seq insert is assembled into BamHI-linearized insulator-seq acceptor library by Gibson assembly (step 7). (O) Product of cloning step 7 – insulator-seq reporter library.

Figure 6

Insulator-seq library preparation overview (modified from Tonelli et al.) (A) DNA-seq library preparation from a plasmid pool (step 13). (B) RNA-seq library preparation from total RNA of transfected S2R + Drosophila cells (step 14).

Figure 7

Bionalyzer trace showing size distribution of the DNA PCR2 product (A) Bioanalyzer trace represented as a gel. (B) Bioanalyzer trace represented as an electropherogram. Blue vertical lines show the optimal size range.

Figure 8

RNA-seq Bioanalyzer trace (A) Bioanalyzer trace represented as a gel. (B) Bioanalyzer trace represented as an electropherogram. Arrows indicate RNA-seq fragments that have a single or a double barcode. Blue vertical lines show the optimal size range.