GSM5818687: 4T1Mock2; Mus musculus; RNA-Seq
The RNA was extracted according to the manufacturer's instructions. As a last step of the extraction procedure, the RNA was purified with the RNeasy Mini-kit RNA sequencing was performed by adapting the technology of SCRB-Seq (Bagnoli JW et al., Nat Comm, 2018) to allow for the high cost-efficient multiplexed transcriptome characterization. Briefly, cells were collected in cell lysis buffer and poly-(A)+ RNA were purified using the Dynabeads mRNA DIRECT Purification Kit (ThermoFisher Scientific). poly-(A)+ RNA were annealed to a custom primer containing a poly-(T) tract, a Unique Molecule Identifier (UMI), and a sample barcode. Retrotranscription using Template-switching oligonucleotides (TSO) was then used to synthetize and amplify 3'UTR enriched cDNA, resulting in barcoded cDNA fragments. Library preparation was performed using the Nextera XT library preparation protocol which introduces i5-P5 and i7-P7 structure for massive parallel sequencing. Quality control was performed following pre-amplification RT and library preparation to ensure quality and length accuracy, as well as to equilibrate sample pooling. Libraries were then circularized and sequenced using an DNBSeq-G400 sequencer (MGI), using the MGIEasy Circularization Kit (MGI). 5-10 million pair-end reads (100 bp) were sequenced for each sample. Raw sequences were called using Zebra caller (MGI) and demultiplexed using Cutadapt. RNAseq was carried out at the Genomics Unit of the Center for Applied Medical Research (CIMA, Universidad de Navarra).