GSM5552398: Mock CoLoC-seq replicate #2 sample 1.3 µg/ml RNase A; Homo sapiens; OTHER
Crude mitochondria were purified from mechanically disrupted cells by differential centrifugation and treated with varying concentrations of RNase A, as specified in sample descriptions. Mock CoLoC-seq samples had been lysed with β-dodecyl maltoside prior to RNase treatment. Cap structures were removed with RNA 5'-pyrophosphohydrolase. The 5'-adapter was ligated to 5'-phosphorylated ends. Then the 3'-adapter was ligated to 3'-hydroxyl ends. The first-strand cDNA synthesis was performed with M-MLV reverse transcriptase with a 3'-adapter-annealing primer. The resulting cDNA was PCR-amplified (15 cycles) with a high-fidelity DNA polymerase and barcoded TruSeq primers. cDNA was purified with the AMPure XP kit, fragmented, end-repaired, and subjected to another round of adapter ligation and PCR amplification. The cDNA samples were pooled equimolarly and size-selected on an agarose gel in the range 10-to-220 nt (excluding the flanking sequences). The pool was sequenced on an Illumina NextSeq 500 instrument (75-nt single-end reads). Single-end RNA-seq selecting against RNase A-cleaved transcripts.