title: GSM9118553: mixed samples, MACseq-1; Homo sapiens; OTHER
library_construction_protocol: Lysates were generated by incubating cell pellets in MAC-seq lysis buffer and incubation at room termperatue with agitation for 15 minutes. Cell lysate was transferred into each well of a new 96-well plate previously prepared with 1µl of 10 nM well-specific RT MAC-seq primer and 7.5µl RT mix; the RT mix contains a TSO primer and external ERCC RNAs for normalization. The mixture was incubated for 2hr at 42C 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 were done with DNA Clean & ConcentratorTM-100 (Zymo Research), and RNAClean XP (Beckman Coulter) and each plate were eluted in 22µl nuclease-free water. The purified cDNA was pre-amplified with KAPA HiFi HotStart ReadyMix (Roche), and MAC-seq PreAmp PCR primer and the quality checked on a D5000 Screentape (TapeStation, Agilent). One barcoded library was prepared per plate using TD buffer and TDE1 enzyme (Illumina) for tagmentation and KAPA HiFi HotStart Ready Mix (Roche) and custom primers (MAC-seq P5 PCR and MAC-seq Indexing Mix) for amplification. Libraries were purified with DNA AMPure XP (Beckman Coulter), quality checked on a DNA1000 tape (TapeStation, Agilent) and quantity verified by qPCR. Two indexed libraries were sequenced on a NextSeq 500 instrument (Illumina) using a custom sequencing primer (MAC-seq Read primer) and a High Output Kit v2.5 75 Cycles (Illumina) with paired-end configuration (25 base pairs for read 1 and 50 base pairs for read 2).