title: GSM8627549: K562,E50; dCas9-KRAB,1; Homo sapiens; RNA-Seq
library_construction_protocol: High-throughput bulk RNA-seq plates were prepared by adding 1 μl of 7.5 ng/μl CEL-seq2 primers(Hashimshony, Senderovich et al. 2016) to 96-well plates. These primers contain sample-specific barcodes that were used to barcode and amplify the mRNA from each sample. After combination of cell suspension with the sample barcode, a mixture of dNTP's and synthetic ERCC spike in molecules were added to the plates using a liquid handling platform and cells were lysed at 65 degrees Celsius. Then, a Reverse Transcription mix with Superscript II enzyme was added to barcode and reverse transcribe the mRNA from all samples to cDNA. After incubation at 42 degrees Celsius, second strand synthesis was done by adding a mixture of DNA polymerase, ligase and RNAseH enzyme, after which material from all samples was pooled and purified. Next, material from all samples was frozen and stored, and part of it was amplified using In Vitro Transcription. The resulting amplified RNA was prepared into sequencing ready libraries following the SORT-seq library preparation protocol(Muraro, Dharmadhikari et al. 2016). Sequencing libraries were indexed with Illumina Truseq small RNA primers.