title: GSM9104140: biorep2 KRT10sorted low batch2; Homo sapiens; OTHER
library_construction_protocol: Total RNA was extracted using the RNeasy Plus kit (QIAGEN, 74136). To extract DNA, cells were resuspened in 200-400 µl of lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS), and heated at 65 °C for 10 min with shaking to reverse formaldehyde cross-linking. Samples were cooled and 4 µL 100 mg/mL RNAse A (Qiagen, #19101) was added. Samples were mixed and incubated at 37° for 30 minutes with shaking. After RNAse digestion, 20 µL proteinase K (20 mg/mL, ThermoFisher AM2546) was added and samples were incubated for 2 hours at 37°, then 95° for 20 minutes (no shaking). DNA was purified using Ampure/SPRI beads. Specifically, 1.8X sample volume of beads was added, mixed by pipetting and incubated for 4 minutes before loading on a magnet, incubating for 5 minutes and discarding the supernatant, then washing twice with 80% ethanol, air-drying the beads for 5 minutes and eluting in 100 µL 10 mM Tris pH 7.5. A typical yield was ~4-5 µg DNA per million cells. PrimeStar (Takara, #R045B) PCR reactions in 100 µL reaction volumes were performed with 500 ng genomic DNA and 20 pmol each primer (GGW130/GGW209) per reaction. The PCR program was 98° for 1 minute, then 5 cycles of 98° 10 s, 56° 10 s, 72° 15 s, and a final 72° for one minute at the end. A Zymo PCR cleanup kit (#D4013 or #D4029 for large volumes) was used to combine the PCR reactions, eluting in 100 µL of the kit's elution buffer. A SPRI clean-up was then performed to remove primers. Specifically, 1.1X bead volume SPRIselect beads were added to the reactions, pipet mixed 15 times, incubated at room temperature for 5 minutes, placed on a magnet, supernatant removed, washed twice with 200 µL 80% ethanol, air dried for 2 minutes, removed from the magnet, pipet mixed 15 times with 46 µL 10 mM Tris pH 7.5, incubated for 2 minutes at room temperature, placed again on the magnet, and 45 µL transferred to a new tube. This purified product was then input to a second PrimeStar PCR reaction in 100 µL volumes to attach the full sequencing primers and barcodes. 20 pmol of each primer (TruSeq adapters with dual i5/i7 indexing) were combined with 50 µL PrimeSTAR, 43 µL water and 3 µL 33X SYBR dye. The PCR program was the same as the first, except without the final 72° elongation for one minute, and samples were stopped after 20 cycles based on the amplification trace. PCR product was then run on a 2% agarose gel and gel purified before sequencing.