title: GSM7158615: MACseq_K562_6hr_Plate2; Homo sapiens; OTHER
library_construction_protocol: K562 cells (5 x 10^3) from each well were aliquoted into a separate 96-well plate, washed in ice-cold PBS twice and centrifuged (500g at 4°C for 4minutes). Supernatant was removed and cell pellets were frozen at -80°C. In detail, 17 μl lysis buffer were added into each well of a 96-well plate containing cell pellets and incubated at room temperature for 15 min under agitation (900 rpm). 12.5 μl of cell lysate were transferred into each well of a new 96-well plate previously prepared with 1 μl of 10 nM well-specific RT MAC-seq primer and 7.5 μl RT mix; the RT mix contains a TSO primer and external ERCC RNAs. The mixture was incubated for 2 hours at 42 C to create well-barcoded full length cDNA and then all the wells of a plate were combined into a single tube. Concentration and clean-up was done via column purification and RNAClean XP beads and each plate eluted in 22 μl nuclease free water. The purified cDNA was pre-amplified with KAPA HiFi HotStart ReadyMix and MAC-seq PreAmp PCR primer and the quality checked on a D5000 Screentape. One barcoded library was prepared per plate using TD buffer and TDE1 enzyme for tagmentation and KAPA HiFi HotStart Ready Mix and custom primers (MAC-seq P5 PCR and MAC-seq Indexing Mix for amplification. Libraries were purified with DNA Ampure XP beads, quality checked on a DNA1000 tape and quantity verified by qPCR. 3'mRNA-seq