GSM9513516: Drug-seq library of CRISPR edited PBMC-derived induced microglia-like cells; Homo sapiens; RNA-Seq
96-well multiplexed libraries (DRUG-seq) were prepared using the MERCURIUS™ DRUGseq kit (Alithea Genomics, 10841) as per manufacturer instructions. Briefly, cells were washed with 100 µL Dulbecco's PBS (Gibco, 14190-144) per well, lysed at 4°C with 20 µL/well 1x Cell Lysis Buffer, and clarified by centrifugation (300 × g, 5 min, 4°C). Up to 20 µL supernatant was transferred to a new 96-well PCR plate and stored at -80°C. For reverse transcription, lysates were thawed and 10 µL was added to wells containing dry barcoded oligo-dT primers (Alithea Genomics, 10513). After resuspension, 10 µL RT Master Mix was added and reverse transcription performed (30 min 50°C, 10 min 85°C, hold at 4°C). Firststrand cDNA was pooled, mixed with 7x DNA binding buffer (Zymo, D4003-1-L), and purified via Zymo-Spin IC column (C1004-50), eluting in 20 µL nuclease-free water. Excess oligo-dT was digested from 17 µL eluate using Exonuclease I, incubating 30 min at 37°C, 20 min at 80°C, and cooling to 4°C. Second-strand synthesis was performed immediately after (addition of 7 µL Second Strand Synthesis reaction mix, 20 min at 37°C, 30 min at 65°C, hold at 4°C). Second-strand cDNA was purified with CleanNGS beads (Bulldog Bio, CNGS005; 0.6x ratio) using a PCR Strip Magnetic Separator (Permagen, MSR812). cDNA concentration was measured using Qubit 1X dsDNA HS Assay Kit (Invitrogen, Q33231); 50–60 ng cDNA was tagmented in a 20 µL reaction (with 4µL of provided Tagmentation Enzyme Buffer) for 7 min at 55°C, then purified with 0.6x CleanNGS beads. The resulting library was pre-amplified with provided kit UDI adapters (10 cycles: 98°C 10 s, 63°C 30 s, 72°C 1 min), then purified twice with 0.7x CleanNGS beads and eluted in 20 µL. Final library quality and concentration were measured on an Agilent 5300 Fragment Analyzer with the HS NGS Fragment Kit (DNF-474-0500). Libraries were sequenced on a NovaSeq X Plus 1.5B flow cell (Read 1: 28 cycles, i7/i5: 8 cycles each, Read 2: 90 cycles).