title: GSM3590733: Patient5_Diagnosis_4; Homo sapiens; RNA-Seq
library_construction_protocol: Bone marrow samples were thawed and processed rapidly. Cells were washed in PBS twice, then stained with antibodies (BD Pharmingen, Franklin Lakes, New Jersey) for leukemic stem cell markers (CD34, CD38, CD90, CD45RA, Lin) and viability (7-AAD) and incubated in the dark for 20 minutes at room temperature. Cells were washed once and resuspended in 3%FBS/PBS. 2μL of RiboLock RNase inhibitor (40U/μL; Thermo Scientific, Waltham, Massachusetts) was added to the cells to be sorted. The protocol for single cell RNA sequencing was performed as per Picelli et al (SmartSeq2; Nature Methods 2013). Briefly, 4 ul of lysis buffer containing 0.2% Triton X-100 solution, 1 ul of dNTP mix (KAPA Biosystems, Wilmington, Massachusetts), 1ul of 10uM 5' anchored oligo-dT oligonucleotide (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′; Sigma Life Science, St. Louis, Missouri) and 1ul RNase inhibitor were added to each well of the 96-well plates. Using a BD FACSAria cell sorter the AML stem cells were identified as the population that was CD34+, CD38 dim, CD90-, CD45RA+, lineage-, and 7-AAD-. Samples were the incubated at 72°C for 3 min in a thermocycler and immediately placed on ice. Samples were then spun down and 5.7ul of RT mix (100U PrimeScript (Takara Biosciences), 10U RNase inhibitor, 1X Primerscript first strand buffer (5X), 5mM DTT, 1M Betaine, 1uM template switching oligo (5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′)) was added to each well. The 96-well plate was spun down and incubated at 42°C for 90 min, followed by an additional 10 cycles of 4 min at 42 °C. 1X KAPA HiFi Hotstart ReadyMix was then added along with 0.1 uM PCR primers (5′-AAGCAGTGGTATCAACGCAGAGT-3′) and the PCR was performed for 22 cycles. PCR purification was performed using Sera-mag SpeedBeads (1:1 ratio of beads: cDNA mixture; Thermo Scientific, Fremont, California). PCR product size distribution is checked using an Agilent high-sensitivity DNA chip, with a good library classified as being devoid of fragments <500bp and with a peak around 1.5-2kb. Library preparation was performed as per manufacturer instructions by Illumina Nextera XT DNA sample preparation kit (Illumina, San Diego, California). Briefly, the tagmentation reaction was carried out on 1 ng of cDNA at 55 °C for 10 min. The amplification of adapter-ligated fragments was performed using Nextera PCR master mix, index primers and barcodes were added to identify each cell for 15 cycles. The PCR products from each cell were then pooled together and purified using Sera-mag SpeedBeads (0.6:1 ratio). A final quality check of the cDNA library was performed using an Agilent high-sensitivity DNA chip, obtaining a broad peak between 300-800bp.  Single cells were sequenced using 100bp paired end sequencing on the Illumina HiSeq 2500.