title: GSM9323072: CD34+ Female cells, shUBA1-CDK16, harvested at Day 3 of myeloid differentiation; Homo sapiens; RNA-Seq
library_name: GSM9323072
library_construction_protocol: CD34⁺ hematopoietic stem cells derived from a female human donor were thawed and pre-stimulated for 4 days in stem cell expansion medium supplemented with SCF, FLT3L, TPO, and IL-3. After pre-stimulation, cells were transduced with lentiviral vectors encoding shUBA1–CDK16 or shGFP control. Transduced cells underwent 3 days of puromycin selection to enrich for knockdown populations. Myeloid differentiation was initiated by transferring cells to differentiation medium supplemented with human GM-CSF and CC100 myeloid differentiation supplement. Cells were differentiaited for 3 days under standard conditions. On day 3 of differentiation, 1×10⁶ viable cells were collected  and frozen in CryoStor (StemCell Technologies) freezing medium. After successful confirmation of UBA1–CDK16 knockdown, cryopreserved samples were submitted for single-cell RNA-sequencing analysis. The library construction for the 10x Genomics GEM-X Single Cell 3' Gene Expression wasdone as per manufacturer's protocol. Viable single cells were partitioned into nanoliter-volume GEMs (Gel Bead-in-Emulsions) alongside Gel Beads and reaction reagents using the 10x Chromium instrument . Within these GEMs, a Reverse Transcription (RT) reaction took place, where poly-A mRNA transcripts were captured by poly(dT) oligos on the bead, which led to the synthesis of cDNA that was uniquely tagged with a Cell Barcode and a UMI (Unique Molecular Identifier). Following the disruption of the GEMs, the barcoded cDNA was purified and subsequently pre-amplified via PCR to ensure sufficient starting material. Finally, this amplified product underwent enzymatic fragmentation, end repair, and adapter ligation to incorporate the requisite Illumina sequencing adapters (P5/P7) and sample indices (i7/i5), yielding the finalized library structure which was then checked for quality and sequenced on Illumina platform.
identifiers.namespace: GEO
identifiers.id: GSM9323072_r1