title: GSM6764997: Donor Aged1, BMMCs, mitochondrial DNA , sample 10; Homo sapiens; OTHER
library_construction_protocol: Bone marrow was collected from healthy young or aged donors. Bone marrow aspirate was diluted with an equal volume of Wash Buffer (PBS, 2% FBS, 1mM EDTA). Ficoll medium was added to SepMateTM (Catalog #85460) tubes and then the diluted bone marrow sample was layered on top followed by centrifuging at 1200 g for 20 minutes at room temperature. The top layer, containing the mononuclear cells, was transferred to a new tube, which was then filled up by Wash Buffer. The mononuclear cells were centrifuged at 300 g for 8 minutes. The supernatant was discarded, and the cells were washed twice and resuspended in Wash Buffer for further enrichment or in Freezing buffer (10% DMSO in FBS). Starting with the BMMCs isolated from the previous step, we enriched CD34+ cells following EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Catalog #17896). Briefly, EasySep™ Human CD34 Positive Selection Cocktail (Catalog #18096C) was added to BMMCs suspension to 100 ul/ml and incubated at room temperature for 10 minutes. EasySep™ Dextran RapidSpheres™ (Catalog #50100) was vortexed and added to each sample to 50 µl/ml.  The mix was incubated for 3 minutes at room temperature. Next, Wash Buffer (7 ml) was added to the tube, The cells were then washed 4 times in "The Big Easy" EasySep™ Magnet (Catalog #18001). Finally, cells were resuspended in Wash Buffer and were centrifuged at 300g for 10 min. The CD34+ positive cell pellet was then resuspended in a freezing buffer (10% DMSO in FBS).  To further enrich the hematopoietic stem cells (HSCs), an aliquot of the enriched CD34+ cells were stained by one of the following two antibody panels. 1) CD34 PerCP-Cy5.5 (Catalog #347222); CD45RA Alexa Fluor 488 (Catalog #304114); CD90 PE-Cy7 (Catalog #561558); and  DAPI (Catalog #D1306) as viability dye. Or 2) CD34 BV421 (Catalog #562577); CD45RA-APC-H7 (Catalog #560674); CD90 PE-Cy7 (Catalog #561558), and 7-AAD as viability dye(Catalog #559925). The cells were further sorted using BD FACSAriaTM for CD34+CD45RA-CD90+ to enrich HSCs. BMMCs, as well as enriched CD34+ and CD34+CD45RA-CD90+ cells were cryopreserved. After thawing, cells were immediately processed for experiment as soon as possible without any culturing. RNA lib and ATAC lib prep:  After tagmentation, cells were immediately processed for GEM generation & barcoding following 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Manual  (version CG000338 Rev A, 10x Genomics) with Step 2. Next, the Post GEM Incubation Cleanup and Pre-Amplification PCR were performed following the same User Manual with Step 3 and Step 4. The pre-amplified library was eluted in 100ul EB buffer instead of 160ul. For RNA library prep, we took an aliquot of 35 ul pre-amplified library and followed the same User Manual (version CG000338 Rev A, 10x Genomics) from Step 6 and Step 7. We developed an in-house protocol to generate the massive ATAC library with enough material for both direct sequencing and mtDNA hybridization. For each sample, 8 reactions of the following were performed (each in 50 ul). 5 μl pre-amplified product, 25 μl NEBnext mix (Catalog M0541L), 2.4 μl 10 μM Nextera N70X primer (Supplementary Data 2), 2.4 μl 10X SI PCR primer B (Catalog #PN2000128), 0.5 μl 100X SYBR Green (Catalog #S7563), and 14.7 μl water. The thermocycler program started with 98°C 45 s., followed by the 6-12 cycles of: 98°C for 10 s., 63°C for 30 s., and 72°C for 20 s.; The actual cycle numbers are determined using the amplification curve and the qPCR was stopped when the curve started to reach plateau. The qPCR products (50ul * 8 wells) were combined and purified using SPRI beads (Catalog #B23318) with size selection 0.4X~1.55X. The expected yield is 2,000 ng to 4,000 ng. mtDNA lib prep: We designed 4 sets of DNA probes, with each probe set covering the whole mitochondrial genome and containing 138 biotinylated oligos that are 120 bp long. The 4 sets of probes ((v1, v2, v3, and v4) are staggered by 30 bp with each other and ensure good coverage on junction areas (Supplementary Data 2). The hybridization started with 500 ng ATAC library products for each probe set. In total, 4 independent reactions were performed in parallel and combined at the end (2,000ng input in total). The detailed incubation and wash methods largely followed the Tube Protocol in IDT xGen Hybridization Capture of DNA Libraries protocol.  Briefly, 500 ng input was combined with Cot DNA and purified by SPRI beads, which was eluted by hybridization mix that contains hybridization buffer, hybridization buffer enhancer, one of the probe set, as well as xGen™ Universal Blockers NXT (Catalog #1079584). The hybridization reaction was performed as below: 95C 30 seconds followed by 65C overnight.  The biotin-streptavidin Dynabeads purification was carried out on Day 2 using xGen Hybridization and Wash Kit. The Dynabeads after capturing and washing were used for final quick qPCR amplification (3-5 cycles). The reaction mix is as below: 20ul beads, 25ul KAPA HiFi HotStart ReadyMix (Catalog #KK2601), Primer 2.4ul P5 (Supplementary Data 2), Primer P7 (Supplementary Data 2), 0.5ul 100X SYBR Green (Catalog #S7563). The PCR program started with 98°C 45 s, followed by the 3-5 cycles of: 98°C for 10 s., 63°C for 30 s., and 72°C for 20 s. The final enriched mtDNA was purified by SPRI beads (1.6X) For each sample, all three libraries were sequenced on Illumina NovaSeq S4 300 cycle kit. The sequencing length was as below. Read1: 149nt, i7: 8nt, i5: 24nt, Read2: 149nt. We expected to sequence 20,000~30,000 raw reads per cell for RNA libraries and ATAC libraries, while 50,000~60,000 raw reads for enrich mtDNA libraries.