P13_M10
3' end RNA-seq of Homo sapiens: adult male peripheral blood monocytes
Total RNA was treated with DNase I (NEB) following the manufacturer's protocol. Stranded, ligation-based, digital gene expression RNA libraries preparation was modified from (PMID: 25730492) as follows: 400ng of total RNA was fragmented, dephosphorylated, cleaned (using SPRI beads) and then ligated to a linker1, using T4 RNA ligase I (NEB). Ligated RNA was cleaned, and 16 samples of each subject were pooled together and then PolyA selected (using Oligo(dT) beads) according to the manufacturer's protocol. RT was performed for the pooled, PolyA+ samples, with a specific primer. Then, RNA-DNA hybrids were degraded by incubating the RT mixture with 10% 1M NaOH at 70 0C for 12 minutes. Ph was then normalized by addition of corresponding amount of 0.5 M AcOH. The reaction mixture was cleaned up using Silane beads and second ligation was performed, where the 3'-end of the cDNA was ligated to linker2 fusing T4 RNA ligase I. The sequences of linker1 and linker2 are partially complementary to the standard Illumina read1 and read2 barcode adapters, respectively. Reaction Mixture was cleaned up (Silane beads) and PCR enrichment was set up using enrichment primers 1 and 2. The libraries were 3'-end sequenced (PMID: 28335028) using Illumina NextSeq 500 and the results obtained in fastq format. Reads from technical replicates of samples were combined.
P13_M10
Charite Universitaetsmedizin, Berlin
P13_M10