title: GSM5419060: Experiment5_A_Pool1_10xRNA [BS07509A]; Homo sapiens; RNA-Seq
library_construction_protocol: For genomic DNA, frozen cell pellets were allowed to warm to room tem-perature, and then were resuspended in 385 µL DNA Quick Extract (Lucigen cat# QE09050) and transferred to a 1.5 mL tube. Cells were incubated at 65 °C for 15 min, followed by 98 °C for 5 min. After cooling briefly, 10 µL Proteinase K (Sigma-Aldrich cat# P4850-5ML) was added, and cell lysate was incubated at 55 °C overnight. The following day, 5 µL RNase A (Sigma-Aldrich cat# R4642-50MG) was added, and the cell lysate was incubated at 37 °C for 30 min. Genomic DNA was precipitated by adding 4 µL Glycoblue (Fisher cat# AM9515), 40 µL 3M Sodium Ace-tate, and 1 mL ice-cold 100% ethanol. After incubating at -80 °C for 1 h, DNA was pelleted by centrifugation at 20,000 g for 30 min at 4 °C. The DNA pellet was washed twice with 70% ethanol, and then resuspended in 200 µL Buffer EB (Qiagen cat# 19086). For total RNA, cell pellets containing 1x106 cells each were resuspended in 350 µL Trizol solution (Fisher cat# 15596026) and stored at -80 °C until RNA extraction. Frozen samples were allowed to warm to room temperature, and then 350 µL cell solution was transferred to a Phase-Lock gel tube (Fisher cat# NC1093153). 70 µL chloroform was added to each Phase-lock tube and shaken vigorously, followed by a 2 min incubation at room temperature. Tubes were centrifuged at 12,000 x g for 10 min at 4 °C. Following centrifugation, the aqueous phase was decanted from each tube and transferred to a new tube. 350 µL 70% ethanol was added and mixed well, and the solution was transferred to a Qiagen RNeasy-mini spin column. Samples were centrifuged at 13,000 x g for 15 s, and the flow-through was discarded. 350 µL Buffer RW1 was added to each column, samples were centrifuged at 13,000 x g for 15 s, and the flow-through was discarded. This Buffer RW1 wash was repeated once more for a total of 2 washes. Next, 500 µL Buffer RPE was added to each column, samples were centrifuged at 13,000 x g for 15 s, and the flow-through was dis-carded. This Buffer RPE wash was repeated once more for a total of 2 washes. After the last RPE wash, the column was centrifuged for an additional 2 min at 13,000 x g to remove residual ethanol. Samples were eluted in 40 µL RNase-free H2O. For DNA libraries, unique Molecular Identifiers (UMIs) and the inner portion of the P5 sequencing adapter were added. 20 µg of genomic DNA was digested with PstI (NEB cat# R3140L) for 1 h at 37 °C, and then purified using the Zymo Clean and Concentrate-25 (Zymo Research cat# D4034) protocol. One cycle of PCR was performed with the following conditions: 8 replicate 50 µL reactions were prepared, each containing 500 ng PstI digested DNA, 1x Phusion Hot Start Flex Mastermix (NEB cat# M0536L), and 200 nM of the primer P5_Plasmid_8N/9N/10N, and incubated at 98 °C for 5 min, 60 °C for 1 min, and 72 °C for 10 min. Replicate reactions were combined and then purified using the Zymo Clean and Concentrate-5 (Zymo Research cat# D4014) protocol, eluting the DNA in 20 µL. For RNA libraries, cDNA was synthesized using the Superscript IV First Strand Synthesis kit (Invitrogen), with 5 µg RNA template and 2 µM primer P5_barcode_0N/1N/2N (containing a truncated sequencing adapter) in two replicate reactions per sample. RNA was first incubated with primers and dNTPs at 60 °C for 10 min, then placed on ice for 1 min. The remaining RT reagents were added, and samples were incubated at 55 °C for 10 min, 80 °C for 10 min, and then cooled to 4 °C. Next, 1 µL RNaseH was added to each reaction, and incubated at 37 °C for 20 min. Single-stranded cDNA was purified using the Zymo Clean and Concentrate-5 protocol (Zymo Research cat# D4014), using 7 volumes of DNA binding buffer and eluting in 10 µL Zymo DNA elution buffer. Unique Molecular Identifiers (UMIs) and the inner portion of the P7 sequencing adapter were added to each single-stranded cDNA molecule using 1 cycle of PCR with the following conditions: 2 replicate 50 µL reactions were prepared, each containing 5 µL cDNA, 1x Phusion Hot Start Flex Mastermix (NEB cat# M0536L), and 200 nM of the primer P7_Plasmid_8N/9N/10N, and incubated at 98 °C for 5 min, 64 °C for 5 min, and 72 °C for 5 min. Replicate reactions were combined and then purified using the Zymo Clean and Concentrate-5 (Zymo Research cat# D4014) protocol, eluting the DNA in 20 µL. For inverse PCR (iPCR) libraries, 40 µg genomic DNA was digested with DpnII for 2 h at 37 °C. Digested DNA was purified using the Zymo Clean & Concentrate-25 column protocol, and digestion was verified by running 100 ng DpnII digested DNA out on a 1% agarose gel. Intramolecular DpnII ligation was performed using DpnII digested DNA at a concentration of 5 µg/mL, and T4 DNA ligase at a concentration of 10,000 U/mL. Ligation reactions were incubated overnight at 4 °C, and ligation products were purified using the Zymo Clean & Concentrate-25 column protocol. scRNA-seq expression libraries were generated using the 10x Chromium NextGem Single Cell 3' workflow (10X Genomics cat# 1000128). For experiment 4, an additional sort for GFP-positive cells was performed on day 4, and 5700 cells were collected for scRNA-seq (Supplemental Table S4) while the remaining cells were expanded in culture for an additional 7 d, at which point cell pellets were collected for RNA, DNA, and iPCR libraries. Separate libraries enriched for reporter transcripts were generated from the cDNA produced in the Post GEM–RT Cleanup & cDNA Amplification step of the 10X Chromium Single Cell 3' (v3.1) library protocol using PCR with the following conditions: 8 replicate 25 µL reactions were prepared, each containing 1 µL amplified 10X Chromium Single Cell cDNA, 1x Phusion Hot Start Flex Mastermix (NEB cat# M0536L), 500 nM of the primer P5_Halfsite, and 500 nM of the primer P7_10xSBbarcodeV2_0N, and incubated 1 cycle at 98 °C for 5 min; 8 cycles (sample dependent) at 98 °C for 15 s, 66 °C for 15 s, and 72 °C for 30 s; and 1 cycle of 72 °C for 10 min.