GSM9798073: Parse Biosciences WT sublibrary 7; Homo sapiens; RNA-Seq
12 mL of fresh peripheral blood was collected using vacutainer EDTA tubes, diluted with 12 mL of DPBS supplemented with EDTA (2mM) and FBS (1%), and then gently layered over Lymphoprep (STEMCELL Technologies, USA) in a 50 mL conical tube. Samples were centrifuged at 800g for 15 min at room temperature with breaks off.  After centrifugation, PBMCs in the white foamy layer were carefully transferred to a new tube. After being washed with DPBS twice, samples were then centrifuged at 250g for 10 min, and the pellets were resuspended in cell culture media (RPMI supplemented with 5% human serum). Given their shorter lifespan in cell culture, PMNs were recovered from the red blood cells layer following 1 x RBS lysis buffer digestion procedure and washed twice with 1X PBS and resuspended in cell culture media. Single-cell suspensions were fixed following the Parse Biosciences Evercode Fixation Kit protocol and stored at −80°C until further processing. Fixed cells were rehydrated and pooled into sublibraries according to the Evercode split-pool combinatorial barcoding workflow. Each sublibrary underwent sequential rounds of combinatorial barcoding, including three rounds of in situ barcoding followed by a final indexing step (i5/i7 dual indexing) during library preparation.After barcoding, cDNA was generated and amplified according to the Parse Biosciences Evercode Whole Transcriptome protocol. Final sequencing libraries were constructed using standard Illumina-compatible adapters and quality-controlled prior to sequencing.Sequencing was performed on an Illumina platform, and raw reads were processed using the Parse Biosciences analysis pipeline to assign reads to individual cells based on combinatorial barcode identities. The single-cell libraries were constructed according to the Parse WT kit manufacturer's protocol. 4166 cells per sample (for a total of 100,000 cells from 24 samples) were barcoded using the reverse transcription barcoding approach. Barcoded cDNA was amplified and verified via TapeStation for size distribution following fragmentation, end repair, A-tailing, and adaptor ligation to complete library construction. All libraries were sequenced as paired-end 150-bp reads using the Illumina NovaSeq X Plus 300 platform with a reading depth of 83-104M reads per sample. Libraries were prepared with the whole transcriptome 100K kit from Parse Biosciences, which involves barcoding and then pooling cells from all samples prior to generation of eight sublibraries. Because of the design of the kit, sequencing reads of all samples are distributed across all eight sublibraries. For further details see attached `README.txt` file.
GEO
GSM9798073_r1