The cDNA was amplified with Platinum Taq DNA polymerase (Thermo Fisher, Catalog no. 10966018) using 0.5 μM of a barcoded 3’ PCR-primer and 5’ PCR primer in a 100 μL reaction volume. The following protocol was used: 94°C, 2 min; 94°C, 30s; 60°C, 30s; 72°C, 15s for a total of 30 cycles. Starting from cycle 8, 10 μL of reaction mix were removed every 3 cycles and analyzed in a 2.5% agarose gel. The lowest number of cycles resulting in visible PCR product was chosen and a new 100 μl reaction volume PCR was set up. The result PCR was purified and concentrated using DNA Clean & Concentrator™-5 (Zymo Research, Catalog no. D4013).
Products of a size around 160 bp were isolated using a 3% agarose PippinPrep cassette (Sage Science, Catalog no. CSD3010) on a BluePippin device. After that, if the samples still presented a contaminant band below the expected size (linker-linker byproduct), a new round of PCR and size selection was made. The samples were quantified by TapeStation and sequenced on an Illumina HiSeq 3000 machine as single reads with 50 cycles. Analysis was performed as described previously using PARalyzer34 (link) built into the PARpipe pipeline34 (link) pipeline mapping the reads to human genome hg19.
Adapters and primers sequences used for PAR-CLIP are listed in Table S3.
Products of a size around 160 bp were isolated using a 3% agarose PippinPrep cassette (Sage Science, Catalog no. CSD3010) on a BluePippin device. After that, if the samples still presented a contaminant band below the expected size (linker-linker byproduct), a new round of PCR and size selection was made. The samples were quantified by TapeStation and sequenced on an Illumina HiSeq 3000 machine as single reads with 50 cycles. Analysis was performed as described previously using PARalyzer34 (link) built into the PARpipe pipeline34 (link) pipeline mapping the reads to human genome hg19.
Adapters and primers sequences used for PAR-CLIP are listed in Table S3.
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