Simplyrna cells kit

Automating RNA isolation was a performed by Maxwell RSC using simplyRNA Cells Kit (Promega). Expression levels of IL-10 and CD73 genes were determined by Real-Time PCR, using TaqMan^® assays (Thermo Fisher) and normalized using the 2−ΔΔCt method relative to B2M, and results are expressed as mean ± SD. For each PCR reaction, 5ng cDNA input was added.

Cells were seeded on 60 mm culture plates and grown until ~ 75% confluence, trypsinized, washed twice with PBS, pelleted, and placed on ice. Total RNA was extracted from cells using the simplyRNA Cells kit (cat # AS1390) with Maxwell RSC 16 (Promega) according to the manufacturer’s specifications. RNA QC, along with that of the downstream libraries (below), was performed using a 4200 TapeStation (Agilent). One mg of total RNA was used to construct libraries with the New England Biolabs NEBNext^Ⓡ rRNA Depletion Kit (Cat# E6310X) and Ultra II Directional RNA Library Prep Kit for Illumina (Cat# 7760 L), according to the manufacturer’s instructions. Dual-indexed libraries were pooled and sequenced at VANTAGE (Vanderbilt University Medical Center) on an Illumina NovaSeq 6000 (S4 flow cell) to a depth of approximately 50 million paired-end 150 bp reads per library. Files containing paired-end reads (in.fastq.gz format) were uploaded to the Partek Flow web-based software platform (version 10.0.21). Reads were trimmed and aligned to the GRCm38 (mm10) mouse genome assembly using the Bowtie2 applet. Aligned reads were annotated, and differential expression analysis was performed using the DESeq2 applet. See the “Data Availability” subsection for access to raw and processed data files.

RNAs were co-immunoprecipitated and eluted from the beads as previously specified. RNAs were purified with simplyRNA Cells Kit (Promega). The RNA was then reverse transcribed with random primers and the RevertAid reverse transcriptase (Thermo Fisher Scientific) following the manufacturer’s instructions. The qPCR was performed in a Quantstudio 6 Flex (Applied Biosystems) using PowerUp SYBRGreen Master Mix (Applied Biosystems) with the transcript-specific primers specified on Additional file 2: Table S4. The enrichment of target sequences in each IP was calculated relative to the Input and NI IP negative controls, to determine if the selected mRNAs were targets. Subsequently, the enrichment in CPEB1 relative to CPEB2, CPEB3, or CPEB4 was calculated to determine the preferential enrichment in one group versus the other, using the delta CT method.