GSM5764339: MITO_ctrl_IN_rep2; Homo sapiens; OTHER
HEK293T cells under labeling and unlabeling in 6-well plate were extracted by 1 mL TRIzol reagent following the manufacture protocol after MERR APEX labeling. The extracted total RNA was eluted into ultrapure water.  Total RNA was treated 1 µL DNase I (NEB) to remove residual DNA and incubated at 37℃ for 15 min. Then, RNA was purified by RNA clean and concentrator kit (Zymo). RNA integrity was analyzed by Fragment Analyzer (Agilent). Only RNA with a RQN (RNA quality number) > 8.0 was used for subsequent experiments as INPUT. INPUT samples were enriched by 10 µL Dynabeads MyOne Streptavidin C1 (Invitrogen). RNA afer enrichment was purified by TRIzol reagent and dissolved in 20 µL ultrapure water as ENRICH samples. cDNA libraries for RNA-seq were constructed from ~300 ng INPUT samples and 5 µL ENRICH samples (enriched from 6.5 μg INPUT) using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) with Poly(A) mRNA Magnetic Isolation Module (NEB) selection. PolyA RNA was fragmented at 94℃ for 12 min. cDNA was amplified 11 cycles for INPUT and 15 cycles for ENRICH samples. The prepared libraries were quality-controlled by Fragment Analyzer (Agilent). Good libraries with most cDNA range from 250 bp to 350 bp were sequenced on the Illumina HiSeq X Ten platform at ~10 million paired (2 x 150) reads per library.