GSM8239948: 121_2_down; Homo sapiens; OTHER
On Day 11, cells were collected and sorted using the BD FACSAria™ Fusion cell sorter. Sorting criteria included the top and bottom 25% of cells based on the ratio of GFP to mCherry fluorescence intensity. The CRISPRi screen was conducted under two conditions using cells with either a reference RORC element - GFP reporter or a mutated 77-AG RORC element - GFP reporter, with each condition tested in duplicate. After sorting, genomic DNA was harvested (Macherey-Nagel Midi Prep kit) and amplified using NEB Next Ultra II Q5 master mix and primers containing TruSeq Indexes for NGS analysis. Sample libraries were prepared and sequenced on a HiSeq 4000. The library preparation for CRISPRi/a-v2 involves amplifying sgRNA sequences, which are then sequenced on an Illumina HiSeq 4000 system. The library includes TruSeq indices for demultiplexing post-sequencing. The PCR amplification incorporates Illumina adapters and a unique sample index, facilitating the sequencing of pooled samples in a single sequencing run with a phiX spike-in of 10% to enhance sequence diversity and read accuracy.