title: GSM4471650: K562_SP1-HyPBase_scCC; Homo sapiens; OTHER
library_construction_protocol: Single cell RNA-seq libraries were prepared using 10x Genomics' Chromium Single Cell 3' Library and Gel Bead Kit. Each replicate was targeted for recovery of 6,000 cells. Library preparation followed a modified version of the manufacturer's protocol. We prepared the Single Cell Master Mix without RT Primer, replacing it with an equivalent volume of Low TE Buffer. Gel-in-emulsion (GEM) generation and GEM-RT incubation proceeded as instructed. At the end of Post GEM-RT cleanup, we added 36.5 µl Elution Solution I and transferred 36 µl of the eluted sample to a new tube (instead of 35.5 µl and 35 µl, respectively). The eluate was split into two 18 µl aliquots and kept at –20ºC until ready for further processing. One fraction was kept for single cell calling cards library preparation, while the other half was further processed into a single cell RNA-seq library. To amplify self-reporting transcripts from single cell RNA-seq libraries, we took 9 µl of RT product (the other half was kept in reserve) and added it to 25 µl Kapa HiFi HotStart ReadyMix and 15 µl ddH2O. We then prepared a PCR primer cocktail comprising 5 µl of 100 µM Bio_Illumina_Seq1_scCC_10X_3xPT primer, 5 µl of 100 µM Bio_Long_PB_LTR_3xPT, and 10 µl of 10 mM Tris-HCl, 0.1 mM EDTA buffer. One µl of this cocktail was added to the PCR mixture and placed in a thermocycler. Thermocycling settings were as follows: 98ºC for 3 minutes; 20-22 cycles of 98ºC for 20 seconds–67ºC for 30 seconds–72ºC for 5 minutes; 72ºC for 10 minutes; 4ºC forever. PCR purification was performed with 30 µl AMPure XP beads (0.6x ratio) as described previously. The resulting library was quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D5000 ScreenTape. Single cell calling card library preparation was performed using the Nextera Mate Pair Sample Prep Kit with modifications to the manufacturer's protocol. The library was circularized by bringing 300 fmol (approximately 200 ng) of DNA up to a final volume of 268 µl with ddH2O, then adding 30 µl Circularization Buffer 10x and 2 µl Circularization Ligase (final concentration: 1 nM). This reaction was incubated overnight (12-16 hours) at 30ºC. After removal of linear DNA (following manufacturer's instructions), we sheared the library on a Covaris E220 Focused-ultrasonicator with the following settings–peak power intensity: 200; duty factor: 20%; cycles per burst: 200; time: 40 seconds; temperature: 6ºC. The library preparation was performed per manufacturer's instructions until adapter ligation. We designed custom adapters (Table S5) so that the standard Illumina sequencing primers would not interfere with our library. Adapters were prepared by combining 4.5 µl of 100 µM scCC_P5_adapter, 4.5 µl of 100 µM scCC_P7_adapter, and 1 µl of NEBuffer 2, then heating in a thermocycler at 95ºC for 5 minutes, then holding at 70ºC for 15 minutes, then ramping down at 1% until it reached 25ºC, holding at that temperature for 5 minutes, before keeping at 4ºC forever. One microliter of this custom adapter mix was used in place of the manufacturer's recommended DNA Adapter Index. The ligation product was cleaned per manufacturer's instructions. For the final PCR, the master mix was created by combining 20 µl Enhanced PCR Mix with 28 µl of ddH2O and 1 µl each of 25 µM scCC_P5_primer and 25 µM scCC_P7_primer. This was then added to the streptavidin bead-bound DNA and amplified under the following conditions: 98ºC for 30 seconds; 15 cycles of: 98ºC for 10 seconds–60ºC for 30 seconds–72ºC for 2 minutes; 72ºC for 5 minutes; 4ºC forever. All of the PCR supernatant was transferred to a new tube and purified with 35 µl (0.7x) AMPure XP beads following manufacturer's instructions. The final library was eluted in 25 µl Elution Buffer and quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D1000 ScreenTape.
attributes.tag: GEO Accession
attributes.value: GSM4471650