title: GSM8997057: Drug-seq library of PBMC-derived induced microglia-like cell, batch 1; Homo sapiens; RNA-Seq
library_name: GSM8997057
library_construction_protocol: 96-well multiplexed libraries (DRUG-seq) were prepared using the MERCURIUS™ DRUGseq kit (Alithea Genomics, 10841) following manufacturer instructions. Briefly, cells were washed using 100µL per well of Dulbecco's Phosphate Buffered Saline (Gibco, 14190-144). Cells were then lysed for 10-15 minutes in 20µL per well of prepared and chilled 1x Cell Lysis Buffer at 4°C.  Lysate was carefully removed from the cell culture plate and transferred to a 96-well PCR plate, then spun at 300xg for 5 min in a centrifuge pre-chilled to 4°C. Up to 20µL of supernatant was then removed to a clean 96-well PCR plate for storage at -80°C for up to one month. For reverse transcription, stored lysate was thawed at 4°C, then 10µL of lysate from each well was transferred to a 96-well plate containing dry, well position specific barcoded oligo-dT primers (Alithea Genomics, 10513). Upon resuspension of the oligo-dT primers, 10µL of prepared RT Master Mix was added to each well and reverse transcription was carried according to provided instructions (incubation for 30min at 50°C, 10min at 85°C, then cooling to 4°C). Resulting firststrand cDNA was collected and pooled, mixed with 7x volume of DNA binding buffer (Zymo, D4003-1-L), and purified on a Zymo-Spin IC column (Zymo, C1004-50). Purified first-strand cDNA was eluted in 20µL nuclease-free water. Excess oligo-dT was digested from 17µL of eluate by adding Exonuclease I Enzyme and Buffer and incubating for 30min at 37°C, 20 min at 80°C, then cooling to 4°C. Second-strand synthesis was carried out immediately after by adding 7µL of prepared Second Strand Synthesis reaction mix and incubating for 20 min at 37°C, 30 min at 65°C, and then cooling to 4°C.  Resulting second-strand cDNA was purified using CleanNGS DNA & RNA Clean-Up Magnetic Beads (Bulldog Bio, CNGS005) at a ratio of 0.6x bead slurry to cDNA. Bead purification was carried out using a 0.2 mL PCR Strip Magnetic Separator (Permagen, MSR812). Upon purification, cDNA was measured using Qubit 1X dsDNA HS Assay kit (Invitrogen, Q33231) and 50-60ng of cDNA was tagmented in a 20µL reaction with 4µL of provided Tagmentation Enzyme and 4µL of Tagmentation Buffer. After incubating at 55°C for 7 minutes, the tagmented library was brought to 50µL and purified with CleanNGS beads at a ratio of 0.6x bead slurry to cDNA.  Finally, the tagmented library was pre-amplified with provided UDI adapters included in kit for 10 cycles (one cycle composed of 10 seconds at 98°C, 30 seconds at 63°C, and 1 min at 72°C) total. In preparation for sequencing, this pre-amplified library was purified twice with CleanNGS beads at a ratio of 0.7x bead slurry to cDNA and eluted in 20uL. Samples were split over two different sequencing runs, with at least one replicate of each studied compound on each run. Sequencing resulted in 615 million read pairs for run one and 1016 million read pairs for run two.