title: GSM8690432: PT18 blood stem cell graft; Homo sapiens; RNA-Seq
library_construction_protocol: Tissue - Tissue biopsies were taken endoscopically with biopsy forceps and collected in ice cold complete RPMI 1640 supplemented with glutamine, 1x pen/strep, 10mM HEPES and 10% FBS and processed within 1 hour of collection. The biopsy samples were pooled together, 10-20 biopsies per sample. Epithelial cells were dissociated by transfer into dissociation medium (HBSS w/o Ca2+ Mg2+, with HEPES 10mM, EDTA 5mM) and agitated for 30 minutes at 37°C at 100rpm, and then vortexed for 30 seconds and then filtered through 70mm strainer. The biopsies were then washed with complete RMPI 1640 at room temperature and then transferred to digestion media (HBSS with Ca2+ Mg2+, FCS 2%, DNase I 0.5mg/ml,Collagenase IV 0.5mg/ml) and agitated for 40 minutes at 37°C at 100rpm. After digestion the suspension was filtered through a 70mm strainer and washed with complete RPMI, spun down 5 minutes at 2500rpm and resuspended in 1ml red blood cell lysis buffer and incubated for 10 minutes at room temperature, spun again for 5 minutes at 2500 rpm and then resuspended in RPMI and viability and cell count were determined using AO/PI dye and Nexcelcom automated cell counter prior to further processing for downstream analyses. For samples with viability 70% or less, dead cells were depleted using a dead cell removal kit (Miltenyi Biotec) following the protocol from the manufacturer. After dead cell removal, viability was routinely >85%. Blood - peripheral blood was collected into CPT tubes (BD Vacutainer) and cells were isolated within 2 hours of blood draw. The tubes were centrifuged for 20 min at 1800G at room temperature without break and then the buffy coat layer of PBMCs was collected and then washed with PBS, centrifuged for 15 minutes at 300G. For stem cell graft processing, peripheral mobilized mononuclear cells were obtained from the cellular therapeutic laboratory and were thawed and washed with PBS and centrifuged for 15 min at 300G. CD34+ grafts - Thawed mobilized mononuclear cells were CD34+ selected using an EasySep Human CD34 Selection Kit II (StemCell Technologies) per manufacturers protocol. Single cell isolations were processed using the 10x Genomics Chromium Single Cell 3' v3.1 kit per manufacturers protocol and run on the 10x Genomics Chromium iX instrument. Libraries were prepared using 10x Genomics Library Construction Kit and consisted of single cell isolations resuspended in master mix and loaded into chip with partitioning oil and gel beads to generate gel bead-in-emulsion (GEM), the poly-A RNA in each GEM was reverse transcripted to cDNA containing an ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and 10x barcode. The pooled barcoded cDNA was cleaned with Silane DynaBeads, amplified by PCR and appropriate sized fragments were selected with SPRIselect reagent. Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and sample index were added during library construction. Final libraries were sequenced on the Illumina NovaSeq 6000 at the Duke Center for Genomic Computational Biology at Duke University