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Additional file 5. Supplemental Material Primer Sequences. A list of all oligo-nucleotides employed in this study, using sequence conventions as outlined by IDT, Inc

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Additional file 2: Table S1. A per-sample overview of sequencing metric details that were used to construct Table 1 of the main manuscript

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Additional file 4: Table S3. A Gene Ontology analysis of the pathways ranked by p-value represented by the DEGs detected by TruSeq in the Wild-Type vs. GFAP-IL6 mouse model

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Additional file 3: Table S2. A Gene Ontology analysis of the pathways ranked by p-value represented by the DEGs detected by 3’Pool-seq in the Wild-Type vs. GFAP-IL6 mouse model

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Additional file 1: Figure S1. A Comparison of DEGs detected by TruSeq and 3’Pool-Seq. A) Venn Diagram depicting the DEGs that are detected by TruSeq, and/or 3’Pool-Seq at the indicated cutoffs. B) A histogram showing Mean TPM, transcript length, and absolute log2(Fold-Change) distributions of DEGs detected by TruSeq and/or 3’Pool-seq

Benchmarking and performance.

(A) Genome size and filtered protospacer density for the five species tested. (B) The fraction of protospacers passing filters of off-targeting, efficiency score, and both. The latter are defined as “filtered protospacers”, whose density is shown in (A). Data are displayed as a fraction of the total number of canonical PAM sequences in each genome. (C) The effect on library quality of modifying design variables. Y-axis denotes the percent of target regions, divided by: “successful”, where n = 10 distinct sgRNA pair designs are returned per target; “intermediate” designs, where 0Materials and Methods for details). The first column represents the run performed with default settings, and in each subsequent column one variable is modified (see Table 3 for details).</p

6xCRE mCherry reporter plasmid map: P17_6xCRE mCherry lenti zeo.

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Pooled screening for B2AR.

A. Design of the experiment. The sgRNA library containing 128 sgRNAs was infected into clone HEK293T + dCas9-AID*Δ + 6xCRE mCherry reporter at about 0.05 MOI. Cells were selected with puromycin, and treated with isoproterenol to stimulate B2AR activity. Cell pellets were obtained for genomic extraction before sorting (presort cells) and sorted for the mCherry negative in both populations (the infected with the B2AR library and the non-infected cells). WT HEK293T cells do not contain the CRISPR-X system, and are used here as negative control. B. mCherry fluorescence intensity histogram, showing the cut-off for sorting mCherry negative cells.</p

Genome-wide knockout libraries for entire classes of genomic elements in humans and other species.

(A) An example of paired sgRNAs designed against the upstream ultraconserved element (UCE) and promoter of the human IRX3 gene. IRX3 lies on the antisense strand. The exact target regions are shown in black, flanked by the design regions in green. The ten sgRNA pairs for each are denoted by red bars. Integrated chromatin marks from the ENCODE project [26] are displayed below, in addition to PhyloP multispecies conservation scores [33]. Note the region of elevated conservation corresponding to the UCE. (B,C) Summary of paired sgRNA designs targeting entire classes of genomic elements. In each figure, the left scale and grey bars represent the design performance, as in Fig 2. The right scale and black bars indicate the total number of elements in each class. (B) shows a series of genomic element classes for human, while (C) shows designs for the entire set of annotated microRNA genes in five species. Designs were created with default settings; designs using “DECKO” construction method give identical results.</p

Targeting B2AR as a model GPCR.

A. Western Blot for Cas9 expression levels in three cell lines: Wild-type HEK293T, HEK293T + dCas9-AID*Δ as a pool, and the single cell clone of HEK293T + dCas9-AID* selected for the highest expression of Cas9. Housekeeping protein vinculin is used as reference. Blots were cropped where indicated by the arrow. Full uncropped blots are provided in S1 Fig. B. Clone HEK293T dCas9-AID*Δ was infected with 6xCRE-mCherry reporter system and single cell clones were evaluated. The most homogeneous clone for mCherry expression was selected. mCherry red fluorescence was measured with high content imaging using a cell incubator imaging system. Cells treated with and without isoproterenol were monitored for 30 hours after stimulation. C. mCherry fluorescence intensity of the same single cell clone as panel B, 24 hours after isoproterenol treatment or in its absence. D. B2AR and B1AR mRNA expression 72h after transfection of 10pmol of siRNA. The mean of 4 replicates is shown. E, Left, mCherry fluorescence intensity of cells transfected with control siRNA or siRNA against B2AR. Right, mCherry fluorescence intensity of cells transfected with control siRNA or siRNA against B1AR. In both cases, cells were stimulated with isoproterenol after siRNA treatment. F. mCherry fluorescence intensity of knock-out cells for B2AR, with and without stimulation of isoproterenol.</p