title: GSM9440659: HEK293T, TotalX miRNA(+)/DASH(-); Homo sapiens; RNA-Seq
library_name: GSM9440659
library_construction_protocol: Single-cell total RNA sequencing was performed using TotalX, a modified 10x Genomics Chromium 3′ v3.1 workflow incorporating enzymatic strategies adapted from Smart-seq-total. The protocol enables the simultaneous capture of polyadenylated and non-polyadenylated transcripts through a co-incubation approach integrating 3′ polyadenylation, 5′ capping, and reverse transcription (RT) in a single reaction. RT master mix was prepared by supplementing the standard 10x RT reagents with the addition of following components: E. coli poly(A) polymerase (New England Biolabs, M0276S; final 1 U/µl), ATP (10 mM), Vaccinia capping enzyme (NEB, M2080S; 0.1 U/µl), S-adenosylmethionine (SAM, 2 mM; NEB, M2080S), guanosine triphosphate (GTP, 10 mM; NEB, M2080S), and a custom biotinylated template-switching oligonucleotide (5′ Biotin-ATGGCUCGGAGAUGUGUAUAAGAGACAGUCUrGrG+G; Integrated DNA Technologies). Reagents were mixed immediately prior loading and kept on ice until droplet generation. Cells or nuclei were loaded into the 10x Genomics Chromium controller following the manufacturer's protocol for 3′ v3.1 chemistry. Following reverse transcription, emulsion droplets were broken according to 10x recovery steps. cDNA was then treated with uracil-DNA glycosylase (UDG; NEB, M0280S) to remove dUTSO fragments at 37°C for 50-60 min. This was followed by 5 cycles of amplification using 10x proprietary primers and a custom spike in primer (according to TotalX protocol). pre-amped cDNA (for 5 cycles) was cleaned up using 1.8X SPRI Select (Beckman Coulter, B23317) and cDNA corresponding to ribosomal RNA was depleted by Cas9-mediated cleavage using guide RNAs targeting mitochondrial and cytoplasmic ribosomal sequences (DASH). CRISPR ribonucleoprotein complexes were assembled from Alt-R S.p. Cas9 Nuclease V3 (NEB, 1081059), tracrRNA (IDT, 1072532), and a custom pool of 57 crRNAs (IDT) designed based on Smart-seq-total DASH oligos (Supplementary Table 1). The reaction was incubated at 37°C for 60 min, followed by treatment with proteinase K (Thermo Fisher Scientific, EO0491) for 10 min at 56°C. cDNA was then cleaned up using SPRI Select and amplified for additional 7-10 cycles using 10x proprietary primers and a custom spike in primer. Amplified cDNA was separated into long- and short-fragment pools using SPRIselect magnetic beads (Beckman Coulter, Cat# B23318). To obtain the long-fragment pool, SPRIselect beads were added to amplified cDNA at a 0.6X bead-to-sample ratio, and bound fragments were retained according to the manufacturer's instructions. Unbound fragments in the supernatant were further purified at a 1.2X bead ratio (1.8X final) to isolate the short-fragment pool. The long-fragment pool was processed according to the Chromium Single Cell 3′ v3.1 protocol (10x Genomics), using the Single Index Kit T Set A (PN-1000213/PN-2000240). Short fragments were indexed using custom primers through PCR amplification containing 10x Genomics 3′ v3.1 library amplification mix, SI primer and custom index primers (see Supplementary Table 1) For each 40 μl reaction, the following components were combined: 20 μl Amp Mix, 4 μl SI primer, 1 μl custom index primer (10 μM), 1 μl amplified cDNA (1 ng/μl), and 14 μl nuclease-free water. PCR conditions were as follows: 98°C for 3 min; 12 cycles of 98°C for 15 sec, 62°C for 20 sec, and 72°C for 2 min; and a final extension at 72°C for 1 min. PCR products were purified with a 1.2X SPRIselect bead clean-up and eluted in 30 μl nuclease-free water. The resulting libraries were either split for further purification to remove very short fragments (below 200-250bp) and obtain the small RNA fraction or used for optional miRNA enrichment. The miRNA(+) fragments were further size-selected from the short-fragment pool by enrichment of fragments corresponding to small RNAs (18–30 bp) using the Pippin Prep gel system (Sage Science, 3% agarose), following the manufacturer's protocol. Short and long pools were recombined in desired ratios prior to final library quality control and sequencing. Resulting libraries were sequenced on a NovaSeq X instrument (Illumina) with the following configuration: Read 1, 28 bp; Index 1, 8 bp; Index 2, 0 bp; Read 2, 91 bp.
identifiers.namespace: GEO
identifiers.id: GSM9440659_r1