GSM7665642: B-ALL-Sample-10; Homo sapiens; ncRNA-Seq
RNA was isolated from EVs using miRVana The library was prepared using 10ng RNA input or less with a library normalized for all samples and the NEXTFLEX Small RNA-Seq Kit v3 (Perkin Elmer, NOVA-5132-24) following the manufacturer's instructions. First, QIAseq miRNA Library QC Spike-Ins (Qiagen, 331535) was diluted 1/2 and then added to the samples resulting in a final dilution of 1/42. NEXTFLEX 3' 4N adenylated adapters were ligated to samples using a 1/4 adapter dilution and a 16°C overnight incubation. The adaptor-ligated samples were purified with NEXTFLEX cleanup beads, and excess adapters were inactivated. Next, NEXTFLEX 5' 4N adapters were added to samples using a 1/4 adapter dilution followed by reverse transcription first strand synthesis. Reverse transcribed samples were subjected to a bead clean-up without any size selection. For the PCR enrichment, a different unique dual index (Perkin Elmer, NOVA-5132-24) was added to each sample, and 22 PCR cycles were used. Amplified libraries were purified without any size selection initially; however, due to high molecular weight templates, a final size selection was performed to obtain the final library. The quality of the library was assessed as follows; size distribution was determined with the D1000 assay on the Tapestation (Agilent, 5067-5582 and 5067-5583). The KAPA Library Quantification Kit (Roche, KK4824) was used to evaluate the concentration. Equimolar amounts of libraries were first sequenced on the iSeq 100 instrument (Illumina) using single-end sequencing of 1X100 to assess both library and pooling qualities. Libraries' inputs were rebalanced following Illumina's recommendations to ensure an equal representation of each sample. Libraries were then sequenced using the Novaseq 6000 instrument (Illumina). Samples were loaded on an S1 flow cell, and a single-end sequencing of 1X101 was used.