GSM6165292: RM30 [ATAC-seq]; Homo sapiens; ATAC-seq
Patient samples were FACS-sorted using CD34+ DAPI- gating. Downstream processing using the 10X Chromium Solution platform for single cell ATAC sequencing, with the modifications of the GoT-ChA protocol. Nuclei were subsequently processed according to the Chromium Next GEM Single Cell ATAC Solution user guide (version CG000209 Rev F, 10x Genomics) with the following modifications: 1- During the GEM Generation and Barcoding reaction (Step 2.1), 1 μL of 22.5 μM GoT-ChA primer mix was added to the barcoding reaction mixture. The primers used are GoT-ChA_R1N (locus-specific primer with a Read 1N handle sequence: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG - [22bp locus specific]) and GoT-ChA_IS2 (locus- specific primer with an IS2 handle sequence: AGCAAGTGAGAAGCATCGTGTC - [22bp locus specific]). These primers allow for exponential amplification of the GoT-ChA fragments, relative to the linear amplification of ATAC fragments. 2- During the Post GEM Incubation Cleanup (Step 3.2), 45.5 μL of Elution Solution I is used to elute material from SPRIselect beads. 5 μL is used for GoT-ChA library construction, and the remaining 40 μL are used for ATAC library construction as indicated in the standard protocol. 3 - To generate the GoT-ChA library, two additional PCRs were performed on the 5 μL set aside during Step 3.2. The first PCR aims to amplify genotyping fragments prior to sample indexing and uses P5 (binds the P5 Illumina sequencing handle: AATGATACGGCGACCACCGAGATCTACAC) and GoT- ChA_nested (a nested, biotinylated, locus specific primer with a TruSeq Small RNA Read 2 handle: /5BiosG/CCTTGGCACCCGAGAATTCCA-[22bp locus specific]) primers with the following thermocycler program: 95 °C for 3 min; 15 cycles of 95 °C for 20 s, 65°C for 30 s and 72°C for 20 s; followed by 72°C for 5 min and ending with hold at 4°C. After a 1.2X SPRIselect clean up, biotinylated PCR product is bound and isolated using Dynabeads M-280 Streptavidin magnetic beads (Thermo Fisher Scientific, #11206D). Briefly, beads are washed three times with 1X sodium chloride-sodium phosphate-EDTA buffer (SSPE, VWR, #VWRV0810- 4L), added to the purified PCR product, and incubated at room temperature for 15 minutes. The beads are then washed twice with 1X SSPE buffer and once with 10 mM Tris-HCl (pH 8.0) before resuspending in water. The bead-bound fragments are then amplified and sample indexed using P5 and RPI-X (binds the TruSeq Small RNA Read 2 handle and adds a sample index and P7 Illumina sequencing handle: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGAC TGGAGTTCCTTGGCACCCGAGAATTCCA, “X” denotes user-defined sample index) primers with the following thermocycler program: 95 °C for 3 min; 6- 10 cycles of 95 °C for 20 s, 65°C for 30 s and 72°C for 20 s; followed by 72°C for 5 min and ending with hold at 4°C ATAC libraries were sequenced to a depth of 25,000 read pairs per nucleus and GoT-ChA libraries were sequenced to 5,000 read pairs per nucleus