GSM5573451: MDA-MB-468 cells, Input; Homo sapiens; OTHER
After the washing steps, the whole recovered material was used for RNA purification with TRIzol and cleaned-up using RNA Clean & Concentrator TM-5 columns (Zymo Research, Cat. N°R1015). RNA was eluted in 20 µl of H2O and 4µl were used for reverse transcription using SuperScript™ IV VILO™ Master Mix (Invitrogen) to check enrichment for circ_0076611 vs. LacZ and Input samples. RNAseq was performed on DNase-treated RNA recovered from ChIRP experiments, and from Input samples as well, on the Illumina platform (NextSeq instrument). The RNA of the ChIRP experiments was controlled on a Bioanalyzer with the Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA) to verify the suitable size distribution and to simultaneously quantify the low RNA amounts. The RNA libraries for the RNA sequencing were prepared using the SMARTer Stranded Total RNA Sample Prep Kit - Low Input Mammalian (Takara Bio. USA, Inc). Each ChIRP-RNA-Seq was conducted combining two independent biological replicates. For the input we used 60ng total RNA and performed ribosomal depletion with RiboGone (Takara Bio. USA, Inc) prior to cDNA and library preparation. For the RNA deriving from the ChIRP experiments we used 1200pg for cDNA and library preparation following the manufacturer's instructions. The quality of the resulting libraries was controlled on a Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA). The quantification of the libraries was performed by qPCR. Sequencing was carried out on a NextSeq 500 instrument (Illumina Inc., San Diego, CA, USA), sequencing in paired-end mode 76bp. ChIRP followed by RNAseq