title: GSM6284348: HSC.RNA; Homo sapiens; RNA-Seq
library_construction_protocol: For colonic epithelial cell collection, ten C57BL/6J male mice at 9 weeks of age were sacrificed and cardiac perfusion was performed using normal saline to deplete circulating immune cells and colonic tissue between the rectum and cecum was isolated. Colonic waste content was flushed and the lumen was exposed. Tissue was then placed in EDTA Dissociation Buffer (4 mM EDTA, 10 mM HEPES pH 7.2, 10% FBS) and rotated at RT for 30 minutes. The epithelial surface was then scraped four to five times with a glass coverslip to collect the mucosa and the remaining tissue was minced finely with scissors. The mucosal and muscle wall tissue were each transferred to Advanced DMEM/F12 (Fisher, 12-634-028 ) containing collagenase 0.4 mg/mL (Sigma, C9263-25MG), dispase II 1.25 U/mL (Sigma, D4693-1G), DNase 1 U/mL (Worthington, LS002004), 10 mM HEPES pH 7.2 and 5 µM Y-27632 (MedChem Express, HY-10071). Following an initial 30 minute incubation, both sets of tissue were triturated every 10 to 15 minutes until dissociated fully to single cells. Both sets of tissue were pooled, washed once with PBS and stained with Calcein Red-AM (BioLegend, 425205) for 15 minutes at room temperature. Cells were then stained with anti-Epcam  PE/Cy7 (Thermo, 14-5791-81), anti-CD45 APC-Cy7 (BioLegend, 103116), anti-Ly6G APC (BioLegend, 127613), anti-SiglecF APC (BioLegend, 155507) and DAPI. Live cells were identified as Calcein+DAPI- events. Both epithelial cells (Epcam+CD45-) and non-granulocytic immune cells (CD45+Ly6G-SiglecF-) were isolated using a BD FACSAria by sorting into Advanced DMEM/F12 with 0.2% RNase-free BSA (AmericanBio, AB01243-00050), RNase inhibitor 0.1 U/µL (Enzymatics, Y9240L) and 15 µM Y-27632.   Human bone marrow samples were purchased from AllCells (BM, CR,MNC, 10M). CD34+ selection was performed using CD34 microbead kit (Miltenyi Biotech, 130-046-703) and FACS- purified for long term HSCs (CD45+Lin-CD34+CD38-CD45RA-CD90+). The following antibodies were used for long term HSC sorting: CD45 PE Dazzle 594 (Biolegend, 304050), Lin FITC (Biolegend, 348801), CD34 APC-Cy7 (Biolegend, 343514), CD38 PE-Cy7 (Biolegend, 303516), CD45RA APC (BD, 564442) and CD90 BV421 (Biolegend cat# 328122). About 5,000 FACS sorted long term HSCs were cultured in expansion medium consisting of StemSpan SFEM II (STEMCELL Technologies) supplemented with StemSpan CD34+ expansion supplement (STEMCELL Technologies, 02691), UM171 (STEMCELL Technologies, 72912) and Stemregenin 1 (STEMCELL Technologies, 72342) in 96 well plates coated with fibronectin (Corning, 354409). After two weeks of culture in the expansion media, about 300,000 cells were harvested and profiled using SHARE-seq V2 protocol.  Frozen human Bone Marrow Mononuclear Cells (BMMCs, Allcells) are thawed in a 37 °C water bath for 1 min and transferred to a 15 ml centrifuge tube. 10 ml of pre-warmed DMEM with 10% FBS was added to cells drop-wisely. The cells were spun at 400g for 3 minutes at room temperature. After removing supernatant, the cells are washed twice in 0.5 ml PBS with 0.04% BSA. To deplete neutrophils, the cells were resuspended in 100 μl chilled DPBS with 0.2% BSA and 10 μl of human TrueStain FcX (BioLegend, 422302) and incubated on ice for 10 min to reduce non-specific labeling. The cells were then incubated on ice for another 30 min after adding 0.5 μl of biotin anti-human CD15 antibody (BioLegend, 301913). After immunostaining, 25 μl of MyOne T1 beads were added to the sample to capture the neutrophils for 5 min at room temperature. We then added 900 μl of DPBS with 0.2% BSA to dilute the sample. The sample was placed on a magnet for 3 minutes and 1 ml of the sample was transferred to a new tube while the sample was on the magnet. The cells were ready for fixation and SHARE-seqV2 experiment. The libraries are contructed using SHARE-seqV2 procotol. SHARE-seqV2