Primeflow rna assay kit

MALAT1 and AcGFP mRNAs were labeled using the PrimeFlow RNA Assay kit (88-18009-204 Affymetrix) according to the manufacturer’s protocol. MALAT1 probes were labeled with Alexa Flour 647 (VA1-11317) and AcGFP probes were labeled with Alexa Flour 750 (VF6-14335), nuclei were stained with DAPI. Cells were visualized using the ImageStream®^X Mark II (Amnis) and images were analyzed using image analysis software (IDEAS 6.2; Amnis Corp). Images were compensated for fluorescent dye overlap by using single-stain controls. Cells were gated for single cells, using the area and aspect ratio features, and for focused cells, using the Gradient RMS feature, as previously described36 (link). To calculate the nuclear fraction of each transcript, the intensity of either Alexa Flour 647 or Alexa Flour 750 within the nucleus (as defined by the Morphology mask on the DAPI staining - Channel 7) was measured, and divided by the total intensity for each cell.

Cells from the SC of EAE mice were isolated using the methods described above with LIVE/DEAD^™ staining and fluorochrome-conjugated Abs for cell-surface markers. In situ hybridization for Osm mRNA was performed using the PrimeFlow RNA Assay Kit (Invitrogen) according to manufacturer’s instructions. Cells were analyzed by flow cytometry using Fortessa X20 (BD systems) and results were analyzed using the FlowJo software.

We cloned gRNA libraries purchased from CustomArray (now GenScript) for each of 5 genomic loci (Supplementary Fig. 2). We transduced these libraries (consisting of a single genomic locus and non-targeting gRNAs in the same pool) at low multiplicity of infection (MOI ≈ 0.3) into K562 cells harboring KRAB-dCas9, and selected for transduced cells as previously described^{9 (link)}. In order to limit indirect effects or other changes in expression due to the expression of KRAB-dCas9, we used a dox-inducible system, inducing KRAB-dCas9 expression with 1 μg/ml doxycycline for 48 hours. We used 30 million cells for each screen.
We used the PrimeFlow RNA Assay Kit (Thermo Fisher; Catalog number: 88–18005) according to the manufacturer’s instructions with some modifications. Specifically, we used 10 million cells per reaction (three reactions per screen) and performed five total washes with 35 °C wash buffer following the staining protocol. We stained each sample for the gene of interest with an Alexa Fluor 647 (AF647, “Type 1”) probeset and against a positive control housekeeping gene with Alexa Fluor 488 (AF488, “Type 4”). For most screens we used control gene RPL13A, but because BAX, BCAT2, FTL, FUT1, NUCB1, and PPP1R15A are <700 kb from RPL13A, we used ACTB for these. Probesets used are listed in Supplementary Table 2.